ANNEX 3.2.

Funding Application for Joint Applied Research Projects

PN-II-PT-PCCA-2013-4

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FUNDING APPLICATION FOR JOINT APPLIED RESEARCH PROJECTS ............................................1
0. EXECUTIVE SUMMARY OF THE PROPOSAL ...........................................................................3

1. TECHNICAL AND SCIENTIFIC DESCRIPTION OF THE PROJECT .................................................4

1.1. The project topic and its practical relevance............................................................4

1.2. Project contribution beyond the state of the art .......................................................4
1.3. Project objectives and outcomes...........................................................................10
1.4. Original and innovative contributions of the project ...............................................11
1.5. Inter-, multi-, or trans- disciplinary characteristics 12
2. IMPACT AND DISSEMINATION OF THE PROJECT RESULTS ....................................................12
2.1. Dissemination and exploitation of the project results.............................................12
2.2. Possible applications with market potential ...........................................................13
2.3. Estimated improvements in the quality of life, with respect to current
performance of products, technologies and/or services.........................................13
2.4. Project integration in the development strategy of partner companies...................14
2.5. Intellectual property protection ..............................................................................14
3. CONSORTIUM DESCRIPTION .............................................................................................15

3.1. Project director......................................................................................................15

3.2. Consortium structure.............................................................................................16
3.3. Partner research team leaders..............................................................................17
3.4. Partner team structure ..........................................................................................19
3.5. Consortium complementarities and synergies between partners...........................23
4. PROJECT MANAGEMENT ..................................................................................................23

4.1. Work plan, deliverables and load balancing ..........................................................24

4.2. Coordination and task schedule ............................................................................36
4.3. Available research infrastructure, and its upgrade/development ...........................37
4.4. Manpower allocation .............................................................................................38
4.5. Project budget and partners share ........................................................................41
5. REFERENCES .................................................................................................................41

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0. EXECUTIVE SUMMARY OF THE PROPOSAL

Copy the summary used on the submission website, www.uefiscdi-direct.ro.

(maximum 4000 characters).

This summary will be sent to external peer reviewers during the selection process. It is
recommended to pay special attention to the writing of this summary in order to encourage the
acceptance of the approached external peer reviewers and to allow an appropriate evaluation of
your proposal.

The concept of autoimmune inner ear disease (AIED) was introduced to explain a pattern of
idiopathic and rapidly progressive (weeks or month) hearing loss which affects both ears in
the same time or sequentially, being associated in 50% of cases with vestibular symptoms
and responding positively to corticosteroid treatment. The autoimmune process can be
induced either at the inner ear level in the case of a primary, target organ response, or
outside, gaining access to the inner ear as a secondary immune response.
Up to date, diagnosis in AIED is based on clinical aspects and the positive response of
patients to the administration of corticosteroids in high doses. Sensorineural hearing loss of
autoimmune causes is potentially reversible with proper treatment, which makes the
research aiming to find a specific test for the diagnosis of AIED of high interest both for clinic
and research. Diagnostic testing sustains the clinical diagnostic and the results obtained in
large trials could clarify the role of immunologic testing in the diagnostic of this disease. The
identification of autoantibodies or autoreactive T cells with specificity against the inner ear
could represent specific markers for this condition. Several antigens have been proposed as
markers in AIED, but their pathogenetic role as well as their relevance for the specific
diagnostic remain uncertain.
Therapeutic options are limited at this time, corticosteroids being the only validated treatment
modality. Hearing loss in AIED can be limited by the administration of corticosteroids, but
only 50% of the patients respond positively and an important percent of the initially
responders become non- responders in time. The mechanisms which are involved in this
process are not known in the present.
The heterogeneity of the existent studies for the identification of serologic markers and the
lack of randomized trials to compare therapeutic responses and evaluate long-term results of
therapy are limiting factors in the guidance for clinicians regarding the management of
autoimmune neurosensorial hearing loss.
The proposed study aims to elucidate several aspects of AIED, like its real incidence, either
as a primary entity, or associated in the frame of systemic autoimmune diseases; finding of a
diagnostic algorithm to be used by clinicians which would lead to better therapeutic results;
the characterization of the therapeutic response of patients and recording of residual damage
after one year treatment. These will allow physicians to an early identification of this condition
leading to an early treatment which could reduce the chances of irreversible damage in the
inner ear. In the same time an in vitro study will take place evaluating the potential specific
and nonspecific markers of this disease in all subjects included in the study. To our
knowledge, there is no study to evaluate a large number of potential inner ear markers
together with an intensive study of the immune system involved, all of these correlated with
the clinical evolution of the patients. The results obtained in the study will be used for the
elaboration of a multiplex immunoassay for the assessment of specific markers in AIED.
Diagnosing and treating the inner ear damage of autoimmune etiology in time can prevent
residual damages and therefore the resulting handicap. Choosing the best therapeutic
solution can significantly limit hearing loss in AIED patients and a better social reinsertion.
Our project proposes to bring valuable information to help clinicians to better understand the
physiopathology of this disease, making diagnostic and therapeutic decisions easier, tailored
to the particular features of each patient.

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1. TECHNICAL AND SCIENTIFIC DESCRIPTION OF THE PROJECT

For information only: maximum 10 pages for this section.

The content of this section corresponds to the first evaluation criterion (technical and scientific
quality of the project proposal).

1.1. THE PROJECT TOPIC AND ITS PRACTICAL RELEVANCE

State the project topic, and specify how it is correlated with the thematic of the call.
Underline the practical relevance of the problem to be addressed and solved.

If applicable, describe the project end-product(s), present the expected results, with if
possible appropriate evaluation and success criteria to determine the end-of-project results.
The topic of the present proposal is in the frame of the thematic: 6.1.2. Elaboration of
diagnostic and medical treatment protocols with impact on health and life expectancy. We
consider the present project in perfect correlation with the thematic of the call because it’s
aiming to elaborate and validate standardized protocols for the diagnosis and treatment of
the autoimmune inner ear disease (AIED), which to date are not available in the clinical
practice. This condition is frequently under-diagnosed in the clinic because of the lack of
understanding of its ethiopathogeny, thus adequate treatment is applied late or incorrectly.
This situation often leads to an irreversible damage in the inner ear with a permanent hearing
impairment for an important number of patients which could have benefited of an efficient
intervention if a prompt diagnosis followed by the proper treatment would have been
available.
Based on a well- designed study, by using correct and complex clinical and laboratory
methods, correlating the paraclinical findings with the outcome of therapy, diagnostic and
treatment protocols will be elaborated to be used with each patient with progressive hearing
loss of autoimmune ethiology. Our project will focus on finding those diagnostic tests which
are specific for this disease, eliminating the examinations which are not offering useful
information and thus examination costs will be significantly reduced. The existence of a
specific test for the diagnosis of AIED is of great interest both for clinic and research, having
in mind that this disease is a reversible form of neurosensorial hearing loss. The elaboration
by Partner 2 Apel Laser SRL of a multiple kit for the detection in one test of the specific
markers for AIED represents an important end-product of the research which will make
diagnostic in AIED faster and easier. By producing such an assay, Apel Laser will increase
significantly its activity area and because of the absence on the market of such a test makes
it an important innovation with real chance of success both on scientific and economic plan.
The validation of new diagnostic and treatment protocols in autoimmune inner ear
disease will make possible the correct approach of the clinicians of this condition in order to
ensure that treatment for these patients is initiated in time thus limiting irreversible damages
to the inner ear.

1.2. PROJECT CONTRIBUTION BEYOND THE STATE OF THE ART

Summarize the state of the art and identify the bottlenecks.

Specify the project contribution to the progress beyond the state of the art.
Include the necessary bibliographic references in section 5.
Sensorineural hearing loss (SNHL) is a frequent condition in otology being usually
irreversible. The potentially reversible forms of SNHL can be divided into two subgroups: 1.
Autoimmune inner ear disease (AIED), consisting in multiple episodes of progressive hearing
loss in both ears and 2. Sudden SNHL (SSNHL), usually an isolated, unilateral and single
event. In 30% of these cases, the disorder can be a part of a systemic autoimmune disease.
[1] The pathophysiology of AIED is still not well understood. It implies that inner ear proteins
are recognized immunologically as foreign or non-self. Multiple potential mechanisms have

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been identified that can result in immune-mediated inner ear pathology.
The concept of autoimmune inner ear disease (AIED) was introduced by McCabe [2]
to explain a pattern of idiopathic rapidly progressive (weeks or months) hearing loss involving
both ears either synchronously or sequentially, with vestibular symptoms in 50% of the
cases, that respond therapeutically to corticosteroids. Multiple studies have attempted to
confirm the existence of such an entity, better characterize its clinical picture, and develop
new methods for diagnosis. The most often used definition of AIED is the presence of
bilateral SNHL of 30dB or more at any frequency with evidence of progression in at least one
ear on two serial audiograms performed less than 3 month apart.[3]
Animal studies have proven that the inner ear does in fact have immunity and is not
an immune privileged site as once thought. The endolymphatic sac has been shown to
contain immunocompetent cells even in a normal resting state and seems to be the site
where antigen processing is carried out. It serves to protect the rest of the inner ear from
foreign substances and infectious agents. Experiments have shown brisk immune mediated
inflammatory responses in the endolymphatic sac after direct inoculation with viral
components with preservation of hearing and balance. The cochlea on the other hand is
devoid of immune cells. It has been shown, however, that when an antigen is injected into
the scala tympani, it will eventually diffuse into the endolymphatic sac where it may
potentially illicit an immune response. Experimental evidence has also supported the
existence of AIED. It has been shown that animals immunized with inner ear proteins can
produce modest, transient hearing loss and cochlear inflammation. [4] Despite this evidence
from animal models, the existence of specific autoimmunity within the inner ear remains
controversial. Although candidate antigens including a 68 kDa protein, type II collagen, and
several other antigens have been proposed, a single target antigen of autoimmunity has not
been found yet. This is not surprising considering the fact that the inner ear is comprised of
thousands of different proteins, many of which are intimately involved in auditory and
vestibular functions.
Histopathologic diagnostic which could establish an autoimmune etiology is of course
impossible to obtain with respect to the inner ear of patients with suspected AIED. A few
temporal bones of patients with suspected AIED have been examined postmortem and have
shown osteoneogenesis consistent with past inflammation, but this examination was not
concurrent with the onset of disease.
The clinical picture of AISNHL overlaps with other pathologic conditions. Our
ignorance of the immune mediating pathways, the need of further animal model
experimentation, the variability of laboratory test results and of patient treatment responses
illustrate how poorly we understand this disorder. In 1984, Hughes and colleagues published
a clinical profile for autoimmune hearing loss developed from 15 patients with laboratory-
suggested AIED.[5] A specific test for AIED would be of great clinical and research
interest since AIED is a potentially treatable form of sensorineural hearing loss.
To date, the diagnosis of AIED is based on clinical presentation and the patients’
response to the administration of corticosteroids. Diagnostic testing may support the
diagnosis, and the results provided by multicenter studies should clarify the role of
immunologic testing in the diagnosis of this entity. Treatment options are limited, with
corticosteroids being the only validated treatment option, and methotrexate offering
no significant benefit to patients.[1]
The diagnosis of AIED is still in an experimental phase. Recent studies in the
literature from large referral centers are based on relatively small sample sizes of
patients who fit the criteria for diagnosis of AIED. Diagnosing an autoimmune disorder as
the cause of inner ear symptoms can be difficult. To succeed, a physician must have training
and experience in these disorders. Most otolaryngologists are not trained or experienced in
autoimmune disorders in general, and a rheumatologist trained in autoimmune disorders is
unlikely to be highly familiar with audio-vestibular function. Thus gaps exist in diagnosis and
treatment. In addition, if vestibular symptoms occur as part of a body-wide problem,
simultaneous non-vestibular symptoms may make the diagnosis difficult. Laboratory testing
is of great value when evaluating a patient with a suspected autoimmune disease. The

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results can confirm a diagnosis, estimate disease severity, aid in assessing prognosis and
are useful to follow disease activity. No slam-dunk sort of diagnostic test exists for this type
of ear problem. There are no specific tests for autoimmunity to the inner ear that are both
commercially available and proven to be useful. The heterogeneity in the designs of the
available studies on identification of serological markers for autoimmune disease and
the absence of randomized trials comparing treatment responses and assessing long-
term outcomes are some of the factors accounting for the limited evidence to guide
the clinician in the approach to the diagnosis and treatment of autoimmune SNHL. In
the three decades since the first report of autoimmune hearing loss, research on the
molecular mechanisms underlying this disease and investigation of treatment efficacy have
advanced clinical understanding and practice.
Hughes proposed two most helpful clinical tests for diagnosing AIED: the lymphocyte
transformation test (LTT) and the Western blot immunoassay. [5] These are both antigen-
specific immune tests. The sensitivity and specificity for LTT are estimated to be 50-80%
and 93% respectively with positive predictive values ranging from 56-73% depending on the
disease prevalence in the tested population. When applied to high risk populations (patients
with bilateral rapidly-progressive SNHL), the Western Blot had a sensitivity of 88%, a
specificity of 80%, and an overall positive predictive value of 92%. For these tests to work,
high levels of circulating autoimmune antibodies are ideal. Because the disease waxes and
wanes, testing should be performed during periods of disease activity and before
treatment is initiated.
Currently, tests for AIED are not routinely used except at certain specialized centers
or in experimental trials.
Autoimmunity may be induced either within the inner ear, in a primary end-organ
response or outside, gaining access to the inner ear as a secondary response. Identifying
autoantibodies or self- reactive T-cells of inner ear specificity may serve as evidence
of autoimmunity and could be candidate markers of this clinical disease. Several
antigens have been also proposed as markers of AIED, but their role in pathogenesis as well
as relevance in disease-specific diagnosis remains undefined.
Although it was shown that sera from ASNHL patients contain antibodies capable of
immunostaining human inner ear tissues, the antigen specificity of the antibodies is currently
unclear. Heat shock protein 70 (HSP70) has been implicated as possible target in AIED but
its specificity is questionable since it is also expressed in a variety of tissues and also it failed
to induce hearing loss in immunized mice. [6] An important percent of healthy individuals
have also elevated levels of HSP70.
Other antigens of great potential include the cochlin protein as well as β-tectorin. The
hypothesis of a pathogenic role for either cochlin or β-tectorin is supported by experimental
induction of hearing loss following adoptive transfer of T cell lines specific for these proteins.
Stimulation of splenic cells by cochlin or b-tectorin-specific peptides in that study
demonstrated a Th1-type response, as interferon gamma was the predominant cytokine
produced. Although these findings are of significance in understanding immune pathogenesis
of AIED, the implications of these results in the clinical setting remains untested.
One study, using antigen specific Western Blot analysis of sera from patients and
healthy controls, demonstrated that cochlin rather than b-tectorin-specific IgG antibodies are
more prevalent in patients with idiopathic SNHL, 14% and 2%, respectively. In the same
study cohort, they found HSP70 IgG responses to be significantly higher in prevalence than
either cochlin or b-tectorin-specific antibodies, this being the first study to compare the
prevalence of IgG antibodies to recombinant proteins that are highly expressed in the inner
ear in association with HSP70 antibody responses.[7]
Cochlin is the most abundant protein expressed in inner ear tissues,[8] its expression being
confined mostly to the cochlear and vestibular labyrinth.[9] Cochlin coprecipitates with the
choline transporter-like target antigen recognized by Kresge Hearing Research Institute-3
(KHRI-3) IgG1 monoclonal antibody that induced hearing loss in animals.[10]
Also, the immunization of SWXJ mice with thep131-150 peptide derived from mouse cochlin
resulted in significant hearing loss which could be passively transferred in naïve recipients

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using peptide activated CD4+ T cells.[11] Other studies found elevated levels of cochlin
antibodies in patients with AIED showing cochlin antigenicity. Autoreactive T cells have also
been implicated in ASNHL. PBMCs from ASNHL patients have been shown to elicit
increased inhibition of leukocyte migration, increased proliferation, and have increased
frequencies of IFN-γ–secreting T cells in response to human inner ear homogenate.[12] The
involvement of T cells in ASNHL is supported by animal studies showing cochlear pathology
and/or hearing loss following immunization of guinea pigs with inner ear homogenate.[13]
Hearing loss in AIED can be improved by corticosteroid administration, but only half
of the treated patients are responders and an important percent of these later patients lose
responsiveness over time. The mechanisms controlling responsiveness to coticotherapy are
still unknown. Several studies showed the role played by the IL1 family as regulators of
inflammation in the way different subjects respond to corticosteroids. The results obtained in
several studies suggested that glucocorticoids enhance IL-1R2 expression and control IL-1β
expression possibly explaining the ability of steroids to reverse some sensorineural hearing
declines. Other cytokines, like IFN-γ also inhibit IL-1R2 expression.[14] This could be an
important mechanism that prevents IL-1β mediated inflammation at sites where this process
is poorly tolerated, thus an increased expression of mIL-1R2 in response to corticoids would
have a protective antiinflammatory effect and any suppressor agent of IL1 β release would
represent a viable therapeutic approach[15]. Other cytokines involved in inflammation were
also evaluated by different research groups. TNF-α is a proinflammatory cytokine secreted
by activated macrophages, monocytes, T and B lymphocytes and fibroblasts. One study
found a statistically significant difference in TNF-α levels in the serum of patients with AIED
compared to control subjects [16], while others found lower levels of TNF-α in patients
compared to the control group.[17]
Despite numerous clinical and animal studies, the treatment recommendation for
AIED remains high-dose systemic corticosteroids. It is difficult to study a clinically diagnosed
disease when there are probably many different etiologies causing similar presenting
symptoms and a response to corticosteroids. Another complicating factor is that AIED is a
relatively rare disorder that precludes the examination of large patient numbers in clinical
trials. Other difficulties with clinical research include nonuniform criteria for hearing
improvement and subjective improvement in hearing often poorly correlating with objective
evaluations.[18] A recent study on the audiometric changes in patients with AIED who
responded to treatment offer an audiometric database that may enable greater precision
in judging clinically meaningful parameters for future studies.[19] In addition, few
studies include vestibular symptoms and their changes with treatment in the outcomes.
Further research in AIED may focus on elucidating the etiology of the disease and
distinguishing primary AIED from secondary AIED. Advances in the development of
diagnostic tests and the role of immunologic testing in this disease are also gains that are
reachable in the near term. Appropriate treatment of AIED may provide reversal of
sensorineural hearing loss and may best be served by exploring new treatment
modalities, focusing on intratympanic delivery. Optimization of dosage and frequencies
as well as long-term effects of intratympanic delivery can be further examined in the
treatment of AIED. [20] Corticosteroid treatment, has limited long-term benefit even in
patients that initially responded to the treatment and also has systemic side effects which
make the search for improved therapies very important. Thus, a combined treatment
approach may prove to be effective. When a patient is suspected to have autoimmune
sensorineural hearing loss, a trial of steroids (prednisone or dexamethasone) for 4 weeks is
started. In cases responsive to corticosteroids, for the long-term treatment
cyclophosphamide is used, because of the severe side effects of long-term corticoid
treatment. Methotrexate treatment have been also tried, but it has been shown
ineffective.[21] Other groups treated AIED pacients with ethanercept (Enbrel) [22], infliximab
(Remicade) which are TNFα inhibitors, but these treatments are still not a common practice,
since their effectiveness is not yet demonstrated. None of these drugs represent official
indications for the treatment of AIED. Having a very important immunosuppressive effect
these drugs can also have impact on the responsiveness against infections or tumors.

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Another drawback of these new drugs is their prohibitive cost. As a last resort, when all
treatments are ineffective, cochlear implant is considered a last resort. But cochlear implants
are extremely expensive, and medical treatment should be attempted in each case.
The measurement of soluble antigens or cytokines in serum and plasma is becoming
increasingly important in the study and management of many diseases, and more important,
the measurement of multiple biomarkers may refine risk stratification in clinical settings. As a
result, there is a growing demand for rapid, precise and cost- effective measurement of such
analytes in both clinical and research laboratories. Multiplex arrays provide quantitative
measurement of a large number of analytes using automated formats. They use traditional
immunoassay principles in which high- affinity capture ligands are immobilized in parallel
arrays. The potential advantages of these assays are: the ability to assay independently and
quantitatively multiple analytes simultaneously in small sample volumes; reduce of costs of
experiments: facilitates data analysis; improves assay range and sensitivity. The key concern
in the evaluation of multiplex arrays is the possibility that multiplexing, in itself, results in
anomalies in the quantitation of some of the analytes, as a result of the so- called “matrix
effect”. To avoid this, for a reliable uniplex assay to be incorporated into a reliable multiplex
assay each analyte must be tested for non-reactivity against all the other antibodies to be
used in the manufacturing of the array. Having such an assay that measures simultaneously
the markers of AIED in the serum of patients would be a valuable tool in the diagnostic by
giving the opportunity to have the results rapidly, exactly and cost- effectively.

The proposed study aims to elucidate the real prevalence of autoimmune inner ear
disease, either as a primary condition or as associated with systemic autoimmune diseases,
to find a reliable diagnostic algorithm to be followed by all clinicians in order to obtain better
results, to characterize the patients’ response to therapy and the residual damage after one
year of therapy. These aims will allow clinicians to identify early and therefore treat early the
damages which occur in the inner ear. These studies are important because they shed a light
on the possibility to identify an important complication of autoimmune diseases and therefore
to treat it. Recognizing and treating early the damage of the inner ear as a major organ
involvement may spare the latter residual damage and handicap. The constitution of a serum
sample bank for AIED may help in the identification of other inner ear markers for
autoimmune disease. Last but not least, the description of the best therapy for inner ear
involvement may add to the progression towards limiting of hearing loss and to the further
social reinsertion of the patients involved. The realization of a multiplex assay for the
detection of the validated markers in AIED represents an important achievement of this
project, as there is no commercially available test on the market. By detecting several
markers in a single test, diagnostic in AIED will get easier, faster and cheaper.
Step by step diagnostic approach with the following algorithm
I. History, symptoms
II. ENL exam: otoscopy, otomicroscopy, tuning fork tests, audiograms: pure-tone
threshold evaluation (including AC and BC for 500, 1000, 3000, 4000, 8000 Hz,
speech discrimination, tympanogram and acoustic reflex, otoacoustic emissions,
vestibular exam; checking the spontaneous and evoked nystagmus; head
Shaking test; Halmagyi test; Romberg test; Unterberger test; indication test; nose
finger test; VideoFrenzel examination with light and in darkness;
III. Rheumatologyc examination in the case of presence of signs for autoimmune
diseases including vasculitis. The BVAS score will be recorded.
IV. MRI to exclude retrocochlear lesions, acoustic tumors, multiple sclerosis,
cerebrovascular accidents.
V. Laboratory studies : Complete blood count (CBC), Erythrocyte sedimentation rate
(ESR), C-reactive protein (CRP), Glucose, Cholesterol/ triglycerides, thyroid
hormones, VDRL, HIV, Lyme, antinuclear, antiendothelial, antiphospholipid
antibodies, etc.

Based on the clinical symptoms and the initial results, based on the inclusion and

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exclusion criteria, the following subjects will be enrolled in the study:

Inclusion criteria: uni or bilateral sensorineural hearing loss with more than 30 dB lost on 2
minimum frequencies; sudden or progressive onset; good general health; MRI imaging with
no pathological findings. Exclusion criteria: age; conductive or mixed hearing loss; middle
ear disease; history of barotraumas, head trauma, noise induced hearing loss, meningitis;
positive MRI for vestibular schwannoma; positive FTA (syphilis); positive blood test for Lyme
disease; positive tuberculosis test; past treatment or use of medication or substances to
cause ototoxicity (aminoglycosides, cisplatin, loop diuretics); positive test for HIV; positive for
Hepatitis B or C; presence of demyelinating disease, such as multiple sclerosis; genetic
context (other family members with sensorineural hearing loss).

The control group: will be composed of the same number of healthy outpatients
VI. Initiation of treatment: Patients will be divided into two groups according to
diagnosis: patients suffering from AIED and patients with AIED and associated
vasculitis. The treatment will be initiated with ▪high doses of oral steroids.
▪Hypertensive and diabetes patients will be injected with intratympanic steroids.
▪Vasculitis patients will be treated with cyclophosphamide and
methylprednisolone for remission induction, followed by azathioprine and/or
methotrexate for remission maintenance according to the EUVAS protocol.
VII. Follow-up: All patients will be monitored for treatment response by performing
control audiograms at 1, 3 and 6 month. Evaluation of response to treatment:
repeated audiograms, patients at 1, 3. 6 months. Audiological follow-up will
consist of pure-tone threshold detection, speech discrimination tests, otoacoustic
emissions, tympanogram and acoustic reflex and also the patients, subjective
improvement of hearing.

In parallel with the clinical study the experimental study will be comprised of:

VIII. Evaluation of serum levels of inflammatory cytokines: IFNγ, TNFα, IL-1α,IL-1β

IX. Evaluation of the lymphocyte populations, study of LT CD4+ and CD8+, in AIED
patients compared to healthy subjects;
X. Evaluation of the effect of in vitro exposure of lymphocytes from AIED patients to
recombinant antigen from the inner ear, compared to the behavior of lymphocytes
from healthy subjects.
XI. Detection of antibodies against specific inner ear antigens: Heat shock protein
(HSP-70); Antibodies to Collagen type II and IX, Antibodies to cochlin, Antibodies
to beta tectorin; the validation of specific markers with diagnostic and prognostic
value will represent the basis for the development of a multiplex assay for the
detection of AIED markers;

The results obtained in the clinical and experimental study will used for:

XII. Statistic analysis, evaluation of results, correlations between lab results and
clinical outcome of the therapy
XIII. Elaboration of standardized protocols for the diagnostic and treatment of AIED
and of a multiple kit for the detection in one test of the specific markers for AIED
Conclusions:
Sensorineural hearing loss of immune-mediated origin may be present as a symptom in
systemic autoimmune diseases (Cogan's syndrome, Behçet's disease, Wegener's
granulomatosis, mixed cryoglobulinaemia, systemic sclerosis, systemic lupus erythematosus,
giant cell arteritis, panarteritis nodosa, relapsing polychondritis) or may occur as a primary
disorder without other organ involvement (auto-immune inner ear disease) (AIED).[23]
Although AIED is usually bilateral and rapidly progressive, in some cases it can present
suddenly and unilaterally.[24] The diagnosis of AIED is still predicted based on clinical

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features, while indirectly on positive therapeutic response to corticosteroids. [25] To date
specific diagnostic tests are not available. [26] All studies to detect molecular markers that
have been conducted to date have proven to be irrelevant and have not lead to any
conclusion. The bottlenecks in this area are represented by:
• The extremely high costs of the research requiring intense team work between clinical
specialities and research laboratories.
• Advances in research and treatment are complicated by the unknown physiopathology of
AIED and the lack of a reliable diagnostic test. This project will try to overcome these
difficulties by studying the immunologic mechanisms involved at cellular and molecular
levels, correlating the results with the data coming from the clinical trial.
• AIED is a relatively rare disorder, making difficult the examination of a large number of
patients in clinical trials. Our study will benefit of the teamwork of well-trained specialists
from two clinical settings, the ENL and Rheumatology services of clinics with high
addressability, treating patients coming from the entire region of Transylvania.
• Other difficulties in clinical trials existing so far are represented by non-uniform criteria for
the assessment of hearing improvement. Our study will correlate the objective evaluation
(by audiograms) with the subjective improvement in hearing.
• To our knowledge, there is no study yet to evaluate several inner ear markers studied in
the same patients together with an intensive immunologic study. Our project will bring
valuable information which will help physicians to understand the physiopathology of this
disease making the therapeutic decision easier and also tailored to the patients’ individual
characteristics.
• Due to the fact that there is no clear diagnosis protocol based on the evidence of a specific
molecular marker presence, it is highly difficult to enrol the patients in a study that would
follow the treatment effect in ear autoimmune diseases. These limitations contribute to the
lack of medical consensus about AIED management and stimulate continued research
efforts. Validating a diagnosis and treatment protocol for AIED represents an innovation
compared to the existent data. Establishing the proper correlations between the clinical and
laboratory findings will have important implications for patient care and management of this
disease in the future and it will also reduce costs by the elimination of unnecessary testing
and also improper treatment for the patients which would not benefit from corticosteroid
treatment.

1.3. PROJECT OBJECTIVES AND OUTCOMES

Describe the project objectives and the scientific and technical barriers that will be lifted by
carrying out the project.
State the project outcomes and their comparative contribution with respect to
previous/preliminary achievements.
The main concept of the project proposal is a prospective study on patients with progressive
neurosensorial hearing loss which can be attributed to autoimmune mechanisms, in order to
establish a diagnostic protocol in this currently under-diagnosed condition and also a
treatment protocol based on the evaluation of serologic markers. These protocols could be
an important tool in the management of this condition by the physicians in order to obtain the
best results, with the conservation of hearing in patients
The major objectives of the project are the following:
1. A clinical study, based on an efficient clinical screening in both ENT and rheumatology
services to identify the patients to be enrolled in the study. Including and excluding criteria
will be established so that the studied cohorts will be as uniform as possible. To succeed, a
physician must have training and experience in these disorders. Most otolaryngologists are
not trained or experienced in autoimmune disorders in general, and a rheumatologist trained
in autoimmune disorders is unlikely to be highly familiar with audio-vestibular function. Thus
gaps exist in diagnosis and treatment. By a tight teamwork of ENL and rheumatology

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specialists, a prospective study including a large number of patients will be initiated,
necessary for an increased statistic value of the results of the study. These patients will be
subjected to the same diagnostic algorithm composed of an exhaustive number of
paraclinic tests, and will be treated based on their particularities in three modalities: ▪ high
dose systemic corticosteroids; ▪ intratympanic corticosteroids in patients suffering of diabetes
or high blood pressure; ▪ ciclophosphamide and methylprednisolone in patients with systemic
autoimmune disease.
2. The immunologic study will seek answers regarding the mechanisms which are involved
in the physiopathology of this disease. The patients’ biologic samples will be analyzed and
compared to those prelevated from healthy subjects focusing on the following aspects:
▪evaluation of lymphocytes populations with the determination of CD4 and CD8+ T
lymphocytes numbers; ▪evaluation of the response of lymphocytes to the in vitro treatment
with recombinant inner ear antigens; ▪determination of serologic levels of inflammatory
cytokines: IL-1β, TNFα, IFNγ, IL-1α. These studies will contribute with important
information regarding the immunologic mechanisms which are involved in this condition.
They will also represent the basis for the inclusion of those tests which proved to be
significant as diagnostic tests in AIED. The results obtained will be correlated with patients’
response to treatment.
3. The study of inner ear specific markers will consist of the evaluation of serologic levels
of autoantibodies against specific inner ear antigens: heat shock protein (HSP70), collagen
II, cochlin and β- tectorin. Based on the obtained values and the correlations between their
levels, the evolution of the disease and the response to therapy, the markers which will be
used in the diagnosis will be selected. These markers will also constitute the components
of a detection multiplex assay developed by Apel Laser.
4. Protocol for the diagnostic and treatment of AIED will be elaborated by correlating the
results obtained in the study, and will represent a validated model to be used in clinical
practice, being a valuable tool in the correct management of patients with AIED.

1.4. ORIGINAL AND INNOVATIVE CONTRIBUTIONS OF THE PROJECT

Underline the original and innovative aspects of the project outcomes.

Autoimmune inner ear disease (AIED) is rare, making it difficult to study, but its incidence is
often overlooked due to the absence of a specific diagnostic test. One can speculate that
there might be effective treatments that simply have not been discovered. Based on the
clinical and experimental data, today there is strong evidence that immune mechanisms are
involved in the aetiopathogenesis of inner ear. Patients suffering from autoimmune hearing
loss have many debilitanting symptoms including sensorineural deafness which can lead to
total hearig loss. The originality of this project consists in providing a clear diagnostic
and treatment protocol for AIED which does not exist in the present in the clinical
practice. Therefore these protocols will help clinicians from different specialities to make an
accurate diagnostic based not only on clinical simptoms but also on specific molecular
markers. This type of protocol was nowhere reported to date. The validation of standardized
protocols for the diagnosis and treatment in AIED represents an innovation compared to the
existing data. The establishment of correlations between clinical data and laboratory results
will allow the anticipation of therapy outcome based on serologic markers, having direct
implications in the management of these patients’ care by eliminating unnecessary testing
and inadequate therapy. Based on the results obtained in this study, a multiple array assay
will be developed for the simultaneous testing, from the same sample of several
markers which prove to be significant for the diagnosis of AIED. This would represent a
quick and specific diagnostic test which would eliminate the need for other, costly tests. To
our knowledge there is no such test commercially available, representing another original
aspect of this project.

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1.5. INTER-, MULTI-, OR TRANS- DISCIPLINARY CHARACTERISTICS

Describe the disciplinary components of the project and their correlation within an inter-, multi-,
or trans-disciplinary approach.
Show how the scientific disciplines are interlinked.
A multidisciplinary team from different research areas is involved in this project, allowing
professionals to accommodate larger functional goals and integrate interventions instead of
working on isolated tasks. Teamwork brings together diverse knowledge and skills of very
competent researchers (physicians of different specialties: otolaryngologists, audiologists,
vestibulogists, rheumatologists, biologists, chemists, physicists, engineers, mathematicians)
thereby increasing the chances to obtain valuable accomplishments in the project
and ensuring the cost efficiency of the research. Medical doctors from the otolaryngological
department are experimented in clinical work regarding the diagnosis and treatment of
patients, therefore they will establish the inclusion and exclusion criteria for the prospective
clinical trial studies and will do the follow up observing the responsiveness to the treatment of
patients with sensorineural hearing loss. Also the physicians specialized in audiovestibular
disorders will perform a detailed audiovestibular exam for all the patients. In addition the
otologic surgeons will inject cortison intratympanic to the selected cases. The
professionalism of the doctors from the rheumatology department will ensure the detection of
deafness cases which appear in systemic autoimmune diseases. Highly trained nurses will
be in charge with the harvesting of biological samples and the administration of treatments.
These samples will be transmitted to the partners, which will perform the experimental
section of this study. Experimented immunologists, chemists and cellular and molecular
biology specialists from the research department of the Oncology Institute will conduct the
immunologic study using the most modern tests and benefiting of the most up to date
technologies. The research department staff of Apel Laser will ensure the detection of inner
ear molecular markers using ultimate generation devices and techniques. Each participant,
as a result of being a member of national and international renowned research institute
and/or university, possesses the research infrastructure needed to accomplish their own
tasks. All the results obtained by each partner will be centralized in the projects database,
analyzed, correlated and will offer the basis for new, standardized protocols to be used in the
clinic for patients with AIED and also will provide the basis for the development of a multiplex
array for the detection of AIED markers.

2. IMPACT AND DISSEMINATION OF THE PROJECT RESULTS

For information only: maximum 5 pages for this section.

The content of this section corresponds to the second evaluation criterion (impact and
dissemination of the project results).

2.1. DISSEMINATION AND EXPLOITATION OF THE PROJECT RESULTS

Describe the main dissemination steps and the main dissemination vehicles used in the project.
The validation of new protocols in the diagnostic and treatment of autoimmune inner ear disease
is a new and innovative direction in scientific research. The exploitation and dissemination of the
results is among the objectives of this project, running the entire period of the project involving all
the partners participating in the study. Our dissemination strategy is to identify and organise the
activities in order to promote the widest dissemination of the knowledge resulted from the project
as well as to promote the commercial exploitation. To fulfil these objectives, clear communication
channels between partners and also with the wider community will be set: the internal
communication infrastructure will facilitate the free flow information across the projects’ sites.

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Contact between partners will be maintained by establishing electronic mailing lists including
members of the project from each partner site.
The projects’ web site will represent an important tool for the dissemination of results. The pages
will include: information about the project and its activities, members participating, contact details,
background information, events (seminars, workshops, conferences); instructional materials
derived from the projects activities: methodological guidelines, etc.; news and updates; for the
internal communication the site can also be used for the distribution of documents for the use of
members which can be password protected.
The members of the project will attend conferences, workshops organized in this domain, where
specialists from the target scientific community will contribute with new ideas and brainstorming
about ways to make use of the projects results. Thinking early in the project about the use of the
results will maximize the impact of the dissemination, thus the members of the research teams
will participate to scientific meetings in the theme of the project soon after the start of the project.
The results obtained during the project will be also published in scientific ISI journals which will
increase the visibility of the projects’ results in the scientific community. Some of the members of
the project, who are working on their PhD will use some of the results to finalize chapters of their
thesis.
An important aspect of dissemination of the projects result consists in the identifying of the
beneficiaries of the projects results: a new protocol for the diagnosis and treatment of AIED and a
multiplex assay for the determination of markers of AIED. The beneficiaries will be:
otolaryngologists and rheumatologists; medical students; immunologists; patients with AIED.

2.2. POSSIBLE APPLICATIONS WITH MARKET POTENTIAL

Estimate the further exploitation of the results, including potential commercialization, beyond the
project duration.

The main results generated by this project will be a protocol for the diagnostic and treatment
of patients with autoimmune inner ear disease (AIED); a serum bank with serum collected
from the patients included in this study; a multiplex kit for the detection of AIED markers.
• The elaboration of a reliable diagnostic and treatment algorithm to be followed by all
clinicians will allow clinicians to identify early and therefore treat early and correctly
this debilitating condition thus limiting the irremediable damages which might occur.
All these will result in an significant reduction of costs for the diagnostic and treatment
of these patients by eliminating tests that give limited information for diagnostic;
eliminating treatment options that are ineffective. Also by recovering sooner, costs
with hospitalization and medication will be diminished, and by the social reinsertion of
the patients, further economic benefits will result.
• The constitution of a serum sample bank for AIED is another result which can be
exploited beyond the duration of the project providing samples for further studies in
the field of autoimmune inner ear disease.
• The realization of a multiple detection test for the most important markers of AIED
could be an important result, as there is no commercially available test on the market
for the detection of AIED markers. By detecting several markers in a single test,
diagnostic in AIED will get easier, faster and cheaper making the future product
attractive for the potential beneficiaries.
.

2.3. ESTIMATED IMPROVEMENTS IN THE QUALITY OF LIFE, WITH RESPECT TO CURRENT

PERFORMANCE OF PRODUCTS, TECHNOLOGIES AND/OR SERVICES

Present how the project outcome might contribute to the improvement in the quality of life,
specifying the particular aspects directly related to the project contributions.

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Assess the comparative advantages of the project outcomes with respect to the known
performances of existing products, technologies and/or services.

Hearing loss, tinnitus and balance disturbance represent common symptoms that have
tremendous impact on the quality of life of the patients suffering from AIED. Progressive
hearing loss may lead to total deafness while balance disturbance represent a handicap for
the patients because they cannot conduct their daily routine. Furthermore, some patients
may present disturbing symptoms associated to the systemic diseases as a result of eyes,
lungs, renal involvement which could further decrease the quality of life. The resulting socio-
economic impact is given by the costs for prolonged hospitalization, patients being taken out
of the work environment and expensive treatments that are followed even there is no proven
efficiency. In the present the diagnosis in this disease is difficult, based only on clinical
symptoms which can occur also in other pathologies; this leads to a later initiation of
treatment, when frequently the lesions in the inner ear become irreversible. AIED is an
important diagnosis to consider because it could be a reversible condition in some situations.
Validating a diagnosis protocol and AIED treatment could represent a tremendous gain for
patient rehabilitation. Getting back their hearing and balance will most definitely increase the
life quality of these patients and also their reintegration in society. Even more, patients
suffering of AIED will be correctly showed their way towards the adequate medical unit, and
will be treated from the early stages of the disease, therefore increasing the medical act
quality and improving the standard of life for the patients.

2.4. PROJECT INTEGRATION IN THE DEVELOPMENT STRATEGY OF PARTNER COMPANIES

Briefly state the correlation between the development strategies of partner companies and
the topic/outcomes of the project.
Specify the partner companies’ needs specifically addressed within the project.
Since founded in 2003, SC Apel Laser SRL has highly performed in promoting and
developing laser technology. Registered as a small enterprise SC Apel Laser SRL has
become during the last years the market leader in the field of laser technology in Romania
with a turn-over during 2012 of 2.092.422 EUR from sales of laser equipments and scientific
equipment as distributor of major international manufacturers, such as Coherent Inc. USA,
Horiba Scientific France, EKSPLA Lithuania, with customer portfolio including main research
institutes and universities in Romania and private companies.
At the same time, using its own research infrastructure valued at 990.000 EUR, Apel
Laser SRL is conducting its own research and development in the field of laser technology,
with patented results such as development of laser micro-machining systems, pulsed laser
deposition systems ( PLD ) and magnetron sputtering systems; development of DPSS laser
devices whith VIS and/or IR emission for medical application like LLLT (Low Level Laser
Therapy), high power DPSS laser systems based on frequency doubling processes, high
power industrial laser systems, chillers for high-power lasers, medical devices with laser
diodes; bio-imaging laser systems.
Apel Laser SRL has already conducted and participated in CEEX, PARTNERSHIPS, and
INNOVATION type-research projects which have resulted in patents, technologies and
photonic devices.
By participating in this project, Apel Laser SRL will widen its activity area, getting
involved in medical research and contributing with the development of an innovative assay to
be used as a valuable diagnostic tool. In this respect, Apel Laser SRL has access to the
latest technology available and the staff consists of highly trained specialists with an
important research background.

2.5. INTELLECTUAL PROPERTY PROTECTION

Present the agreement within the consortium for intellectual property protection.

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Specify the main actions for intellectual property protection.

The exploitation and dissemination of results is among the main goals of this project and will
be done in accordance with the international legislation regarding the intellectual property
rights (IPR), this fact being agreed among all the consortium members.
• Each participant is the ownner of the results generated during the project
(foreground).
• The communication of the results among partners will be established in a way that
will ensure the protection of IPR and confidentiality.
• Any dissemination activity regarding results obtained during the project will be
anounced to the other consortium members at least 45 days before the
dissemination.
• When the results will be published, the list of authors must reflect the real contribution
of each author.
• All dissemination activities will ensure the visibility of funding of the project.

3. CONSORTIUM DESCRIPTION

The content of this section corresponds to the third evaluation criterion (quality of the
consortium).

3.1. PROJECT DIRECTOR

The project director should provide a short curriculum vitae (1 page maximum), containing:
- last name, first name, age, career path, current position
- professional experience in the topic of the project
- list of the five most significant achievements related to the project (e.g. peer-reviewed
publications in scientific journals, national/international patents, prototypes,
demonstrators, innovative technologies and services).
- previous projects related to the present proposal
- prizes, distinctions, membership in prestigious international professional associations
Maniu Alma Aurelia, age 45
Career path: 2004-2007: Assistant; MD; ENT Department, “Iuliu Haţieganu” University of
Medicine and Pharmacy Cluj-Napoca Romania; 2007: PhD in Medicine- “The air spaces of
the middle ear “Diploma No. D005303 issued by the Romanian Ministry of Education and
Reserch
Curent position: Lecturer, Ph D, MD: ENT Department, “Iuliu Haţieganu” University of
Medicine and Pharmacy Cluj-Napoca Romania
Professional experience in the topic of the project: Dr. Alma Maniu’s research is mostly
focused on otology, on the etiopahogenesis and new therapies of deafness. Since 2008 her
scientific studies are related to the inner ear sensory epithelia regeneration in cell culture.
She developed a new technique for sampling of the Corti organ from mice and in
cooperation with the Oncological Institute laboratory studied the protective effect of
antioxidants against chemotherapeutic agents’ ototoxicity in the organ cultures of the rat
cochlea. She also has experience in partnership projects, being member in the research
team in two national grants. Currently her major research interest is studying and
understanding the causes of the autoimmune sensorineural hearing loss in order to find the
best treatment modalities. She is involved in collaborative clinic screening work with the
department of rheumatology for early diagnosis of autoimmune ear disease.
Published papers:
1. Rapid progressive bilateral hearing loss due to granulomatous otitits media in Lyme
disease. Maniu A, Damian L. Am J Otolaryngol. 2013 Jan 10. doi:pii: S0196-
0709(12)00258-X.10.1016/j.amjoto.2012.11.009. PMID:23313123

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2. Protective effect of L-N-acetylcysteine against gentamycin ototoxicity in the organ
cultures of the rat cochlea. Maniu A, Perde-Schrepler M, Cosgarea M. Rom J Morphol
Embryol. 2011;52(1):159-64. PMID:21424048
3. Mastoid obliteration with concha cartilage graft and temporal muscle fascia. Maniu A,
Cosgarea M. ORL Journal for Oto-Rhino-Laryngology Head and Neck Surgery
2012;74:141-145 (DOI: 10. 1159/000337093
4. The methodology of harvesting and cultivating in vivo of the middle ear neurosensorial
epithelium in mouse. Maniu A. Romanian Journal of Rhinosinusal Surgery 2007; 5-10
5. Sudden hearing loss surgical treatment. Maniu A, Gocea A, Mociofan E.D, Negru B.
Romanian Journal of Rhinosinusal Surgery nr. 1-2, (2), 2004; 29-38
6. Technical and functional hearing results after unilateral stapes surgery for otosclerosis at
Cluj-Napoca University Hospital. Maniu A, Cosgarea M. Eur Arch Otorhinolaryngol. 2012
Nov 13
7. Mastoid obliteration with concha cartilage graft and temporal muscle fascia. Maniu A,
Cosgarea M. ORL Journal for Oto-Rhino-Laryngology Head and Neck Surgery
2012;74:141-145 (DOI: 10. 1159/000337093)
Grant-Projects
2006-:Grant no 183/2006 CEEX – Study of the inner ear sensory epithelia regeneration
through development of hair cell cultures in mammmals – member in the research team
2008-:Grant no 2620/2008-CNCSIS complex exploratory programs- coordinator of the
project Protective effect of the antioxidants against chemotherapeutic agents ototoxicity in
the organ cultures of the rat cochlea
Speaker at international conferences
Wullstein Symposium: Otology at the interface between research and clinical care: 12.2011-
Wuertzburg, Germany:Antioxidants against Cisplatin ototoxicity in the rat cochlea
28. Politzer Society Meeting Athens Grece 10.2011-Protective effect of L-N-acetylcysteine
against gentamycin ototoxicity in the organ cultures of the rat cochlea.
Membership in prestigious international professional associations
Member of the Romanian Society of Otorhinolaryngologists
Member of Politzer Society The International Society for Otologic Surgery and Scienc
Member of European Academy of Otology and Neuro-Otology
Member of European Society of Pediatric Otorhinolaryngology

3.2. CONSORTIUM STRUCTURE

Briefly describe each partner (CO, P1-Pn) and provide the necessary elements to assess their
qualification in the project ("why who does what"). These elements can be past achievements,
indicators (publications, patents), why the partner is interested in the project, etc. (maximum 0.5
page per partner)
CO – Coordinating Organisation
P1-Pn – Partner Organisation
CO- „Iuliu Hatieganu” University of Medicine and Pharmacy (UMFCN), University of
Medicine and Pharmacy “Iuliu Haţieganu” Cluj-Napoca (UMF) is the oldest higher education
institution with medical profile from Transilvania and one of the most appreciated from the
entire country. UMFCN promotes and supports high quality research and training for medical
studies with a present focused in a broad spectrum of research topics including clinical
studies, functional studies, biomarkers, biotechnology, pharmacology, drug development,
development of novel (noninvasive) diagnostic tools and public health.The Research Center
of the University has a total dedicated area of 1400 sqm, the organization of the Center being
developed as a unit for fundamental and translational research. In the period 2005-2010
were acquired IT and R&D equipment over 5 million euros, equipment that is used mainly for
clinical research. Through the state budget programs and also through European
Commission funds, in period 2010-2011, our university has implemented a number of 149
research grants as coordinator and partner. UMFCN through its mission of education and
research, promotes and supports scientific research by investment in top research

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infrastructure, human resource development, administrative structures to support research
management and by promoting publications and utilization of research results. The
University is ranked first among medical academic schools in Romania as a „research and
education university” having as main middle term strategy „international collaboration and
cooperation with scientists abroad and want to develop research projects in Romania as well
as international”. In the proposed project, the research team of UMFCN will conduct the
clinical study.
P1- Oncology Institute “Prof.Dr.I.Chiricuta” is a medical institution with an important research
department aiming to plan, design and put into practice strategies that will keep the performance
and achievements of the scientific research at high standards. Starting with year 2000, the
research department was equipped with the most modern R&D technology which was developed
throughout the years benefiting of funding from the numerous national and international grants in
which the Institute was involved as coordinator or partner. This participation concretized in the
publication of 203 articles in ISI journals in the last 4 years. The research team of the Cell
Culture, Radiobiology and Tumor Biology Department involved in this project participated in
previous projects regarding neurosensorial hearing loss in collaboration with the ENL department
of the Iuliu Hatieganu University of Medicine and Pharmacy, materialized in several publications
in ISI journals and also presentations at national and internationals scientific meetings. In the
present project, the research team of IOCN (P1) will be in charge of the immunologic study
evaluating immunocompetent cells and cytokines in the serum of AIED patients, compared to the
values in healthy subjects and their correlations with the patients’ response to therapy.
Apel Laser SRL Bucharest
Since founded in 2003, SC Apel Laser SRL has highly performed in promoting and
developing laser technology. Registered as a small enterprise SC Apel Laser SRL has
become during the last years the market leader in the field of laser technology in Romania
with a turn-over during 2012 of 2.092.422 EUR from sales of laser equipments and scientific
equipment as distributor of major international manufacturers, such as Coherent Inc. USA,
Horiba Scientific France, EKSPLA Lithuania, with customer portfolio including main research
institutes and universities in Romania and private companies. In the present project, Apel
Laser will evaluate serum levels of AIED markers compared to healthy subjects, and will
elaborate a multiplex immunoassay including the markers validated by the study.

For all partners, indicate in a table (see below) other on-going projects having possible links with
the proposed research work.

Project name, funding

Partner Name of involved people Start and end dates
institution, grant allocated

CO

P1

P2

Pn

3.3. PARTNER RESEARCH TEAM LEADERS

Each partner research team leader should provide a short curriculum vitae (0.5 page maximum),
containing:
- last name, first name, age, career path, current position
- professional experience in the topic of the project, related to the partner task.

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 - list of three most significant achievements related to the project (e.g. peer-reviewed
publications in scientific journals, national/international patents, prototypes,
demonstrators, innovative technologies and services).
- prizes, distinctions, membership in prestigious international professional associations
Partner 1
Research team leader: Dr. Perde - Schrepler Maria, MD, age 45 years
Career path: 1992- graduated the Faculty of General Medicine of the “Iuliu Hatieganu”
University of Medicine and Pharmacy; 1994-1998- specialist in laboratory medicine; attended
several training courses in the domain of molecular medicine, biomembranes, radiobiology,
functional genomics, cancer biology and therapy, etc.
Current position: research scientist, senior physician in laboratory medicine in the
Radiobiology and Tumor Biology department of the Oncology Institute “Prof.Dr.I.Chiricuta”
since 1999.
She has experience in studying the effects of certain drugs or natural products on cell
cultures of different origins, cellular and molecular mechanisms involved in several
pathologies, using modern research techniques and methods: immunocytochemistry, flow-
cytometry, immunomagnetic cell sorting, ELISA, Western Blot, citotoxicity tests, cell
proliferation tests, apoptosis, DNA damage, cytogenetic tests, DNA extraction.
Project manager on behalf of the Oncology Institute in four national grants.
She participated in two previous grants related to the proposals’ subject, regarding the
sensorial epithelia of the inner ear, one as coordinator on behalf of IOCN.
Publications:
1. Current strategies for the protection, regeneration and replacement of cochlear hair cells,
Perde-Schrepler M, Maniu A, et al, J.Otolaryngol.Head Neck.Surg., 2012, 41(4): 227-239.
2. Protective effect of L-N-acetylcysteine against gentamycin ototoxicity in the organ
cultures of the rat cochlea, Maniu A, Perde-Schrepler M, Cosgarea M, Rom J Morphol
Embryol, 2011, 52(1):159–164
3. Effects of ototoxic drugs on Corti’s explants: experimental study, M.Chirila, A.Maniu,
V.Necula, M. Perde-Schrepler, et al Appl.Med.Inform, 2011, 29(3):25-30.
Diploma of excelence and gold medal – A.Filip, M.Achim, I.D.Postescu, M.Perde-Schrepler,
et al – for pharmaceutical cosmetics with photochemprotective effects - Inventica
International Salon, PRO INVENT, 11th Edition, 2011, Cluj-Napoca
Pending patent (OSIM): Photochemoprotective gel and obtaining procedure)
Membership in prestigious international professional associations: Romanian Society of
Medical Radiotherapy and Oncology, ESTRO
Partner 2
Research team leader: Becherescu Barbu Dan Nicolae, age 27 years
Career path: 03/10/2011 → ; PhD student - Solid state physics; Faculty of Physics,
Bucharest University; 04/10/2009 - 30/06/2011; Master in Photonics, Spectroscopy, Plasma,
Lasers; Faculty of Physics, Bucharest University; 05/10/2009 – 15/06/2011; Master in
Theoretical Physics (Quantum physics, Theory of Relativity and Electromagnetism); Faculty
of Physics, Bucharest University; 03/10/2005 – 04/06/2009 ; Diplomat Engineer;
Technological Physics; Faculty of Physics, Bucharest University.
Training : Horiba Scientific – specialization in Raman systems, SPRi, ellipsometers,
fluorescence spectrometers and XRF; JPK Instruments – AFM and Optical Tweezers –
Advanced Operation / Installation; ANDOR –CCD; EMCCD and iCCD cameras– Learning
the basics;
Projects:
-Project manager on behalf of SC Apel Laser SRL for project PN-II-PT-PCCA-2011-3
Process and device for thi n films deposition in highly ionized pulsed plasma - acronym
PIDESS, contract 174/2012 - Coordinator "A. I. Cuza" University from Iasi
- Project manager on behalf of SC Apel Laser SRL for project PN-II-PT-PCCA-2011-3
Plasmonic 2D optical memory with active chalcogenide glass layer - acronym MEMOPLAS
contract 25/2012 - Coordinator University Politehnica from Bucuresti

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- Project manager on behalf of SC Apel Laser SRL for project PN-II-PT-PCCA-2011-3
Sensor systems for secure Operation of Critical Installations - acronym SOCI - contract
8/2012 - Coordinator National Institute for Laser, Plasma and Radiation Physics from
Bucuresti.

3.4. PARTNER TEAM STRUCTURE

For each partner, indicate the research team structure (research positions, other than the
team leader), in correlation with the allocated task(s).
For the key persons within the research team provide a short curriculum vitae (0.5 page
maximum), having the same structure as the research team leader CV.
CO- research team
Key persons

Violeta Necula age 42

Career path: 2004-2012: Assistant Profesor ENT Department, UMFCN; 2011: PhD in
Medicine “Auditory performance, communication ability and health-related quality of life in
cochlear implanted patients “ Curent position: Lecturer, Ph D, MD, ENT Department, “Iuliu
Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
Professional experience in the topic of the project: The main interest area includes
particularly the ear pathology. Over 10 years experience in clinical activity in the diagnosis of
hearing loss using electro-physiological methods. Member of the cochlear implant team.
In the project, she will perform a proper audiologic examination on all the patients in order to
select the patients who fit de autoimmune inner ear loss criteria for diagnostic. She will also
track patient's response to treatment objectively by improving of hearing on audiogram.
Published papers:
1. Necula V, Cîmpeanu L, Cosgarea M, Boca A, Chira D, Nistor B. Auditory Performances
and Speech Production Outcomes in Cochlear Implanted Children. Studia UBB Psychol.-
Paed., LVI, 1, 2011, pp: 35-46
2. Necula V, Necula SE, Cosgarea M. Importanţa screeningului neonatal în diagnosticul şi
tratamentul hipoacuziei – Obstetrica şi Ginecologia LIX (2011) 123-126
3. Necula V, Cosgarea M, Necula S-E. Health-related quality of life in cochlear implanted
patients in Romania. Int J Pediatr Otorhinolaryngol 2013; 77(2):216-22
Grants-Projects- participated as member in national grants; member of HEAR-RING
Project, sponsored by Orange Foundation
Member of the: Romanian Society of Otorhinolaryngologists, European Academy of Otology and
Neuro-Otology, European Society of Pediatric Otorhinolaryngology

Damian (Man) Laura Otilia, age 45

Career path: 1992- graduate of the “Iuliu Haţieganu” University of Medicine and Pharmacy
Cluj-Napoca;;1993-1998- Rheumatology specialist training, Rheumatology Dept, UMFCN;
1994- EULAR mobility, focused on systemic vasculitis, Rheumatology Dept, Edinburgh,
Scotland; 1996-1999- PhD grant, “Iuliu Haţieganu” UNFCN; 2000: PhD in Medicine-
“Rheumatoid vasculitis “
Her duties in this project will include rheumatology patients recruitment, diagnosis, treatment
and follow-up
Curent position: Rheumatology Consultant, MD, PhD, Rheumatology Department, Cluj-
Napoca, Romania
Professional experience in the topic of the project: focused on collagen vascular
diseases-mostly vasculitides- and rare diseases, on the clinical associations and new
therapies. She also has experience in partnership projects, being member in the research
team in two national grants. Published papers:
1. Rapid progressive bilateral hearing loss due to granulomatous otitits media in Lyme
disease. Maniu A, Damian L. Am J Otolaryngol. 2013 Jan 10. doi:pii: S0196-0709(12)00258-
X.10.1016/j.amjoto.2012.11.009. PMID:23313123

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2. Hemophagocytic syndrome in systemic vasculitis: an underrecognised complication?
Damian L, Cismaru C, Maniu A, Rogojan L, Ghib L, Rednic S, Bojan A: Presse Med 2013,
Apr 20…
3. Catastrophic antiphospholipid vascular involvement in autoimmune rheumatic diseases,
Rednic S, Damian L,“ Bone health and vascular health in chronic inflammation”, Alexandra
Craciun Ed, ed Dacia, Cluj, 2007, 34 – 40
Professional memberships: Romanian Society of Rhjeumatology, Romanian Society of
Pediatric Rheumatology, OSART Romanian Society for Osteoarthrology, Rare Diseases
Foundation, DCVAS.

CO- Research team members

Harabagiu Oana Elena, MD, assistant Professor,: ENT Department, is a young PhD
student studying the theme „Involvement of keratinocytes in the development and
aggressiveness of middle-ear cholesteatoma”; Her task will be to select patients for the trial
and record all results in the patients’ charts.
Cătana Iuliu Vlad, MD, Associate assistant Profesor,: ENT Department, recently graduated
of the PhD studies, is involved in collaborative clinic screening work with the department
of rheumatology for early diagnosis of autoimmune disease. He also has experience in
partnership projects, being a codirector in an international study regarding autoimmunity. He
will provide patients’s enrollment database on working on statistical analysis of the study.
Petri Maria, young PhD student having a scholarship, interested in vestibular peripheral
disorders studying the theme The management and quality of life in patients with vestibular
peripheral disorders. She will provide the vestibular examination of the patients by specific
methods, clinical vestibular examination and follow up.
Ghib Linda, MD, rezident in rheumatology, will be in charge of following the patients’ clinical
evolution.
Popescu Mihaela, head nurse of the E.N.T. Department Emergency County Hospital, Cluj-
Napoca has experience with management of the patients; she will be in charge with the
prelevation of biological samples and treatment administration
Ciursa Gabriela, age 38, nurse, qualified in immunosuppressive drug infusions.
Raducu Loredana Diana- financial administrator
Nistor Mariana- acquisitions

Partner 1- research team

Key persons

Virag Piroska, age 41,

Licensed in Biology; Masters in Cellular Biology; Phd in Biology – “The therapeutical effects
of cytokines in oncological radio- and chemotherapy”; currently principal biologist, head of
the Radiobiology and Tumor Biology Laboratory of the Oncology Institute
Professional experience: study of normal and tumor cells; testing of antineoplastic agents;
identification of predictive molecular and cellular markers for the response to chemo- and
radiotherapy; inflammatory biomarkers in cancer; will be involved in the determination of
serologic cytokines in AIED patients, statistic analysis of results
Relevant published papers:
1. A.Dorhoi, V.Dobrean, M.Zahan, P.Virag. Modulatory effects of several herbal extracts on
avian peripheral blood cell immune responses, Phytother. Res., 20, 2006: 352-358.
2. P.Virag, I.Brie, E.Fischer-Fodor, M.Perde-Schrepler, C. Tatomir, et al, Assessment of
cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for
oxaliplatin resistance, Cell Biochem & Function, 2011 Jul;29(5):351-5.
3. P.Virag, M.Perde-Schrepler, E.Fischer-Fodor, C.Tatomir, et al, Superior cytotoxicity and
DNA cross-links induction by oxaliplatin vs cisplatin at lower cellular uptake in colorectal
cancer cell line. Anti-Cancer Drugs, 2012 Nov;23(10):1032-8.
Previous projects related to the present proposal:

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• Project director of a bilateral cooperation project with Hungary (Capacities, Module III,
2013-2014): „Study of the prognostic importance of some cellular and molecular
inflammatory biomarkers in colorectal and breast cancers”.
- Diploma of excelence in research – Piroska Virag – for remarkable merits in the scientific
reasearch activity of the Oncology Institute „Prof. Dr. I. Chiricuta”, Cluj-Napoca.
Membership in professional associations: Hungarian Academy of Science; Romanian
Society of Medical Radiotherapy and Oncology; National Society of Cellular Biology;
European Organisations of Cancer Institutes

Fischer-Fodor, Eva, Age: 44 years, will perform the magnetic cell sorting of the
mononuclear cells separated from the blood of the patients included in the study. Culture of
the separated lymphocyte populations.
Carreer path: PhD in Chemistry (2008, Babes Bolyai University Doctoral School,
bioorganometallic and bioorganic chemistry), MS in Organic Chemistry and Technology,
Principal Chemist in Clinical Biochemistry
Current position: Research Scientist and Principal Investigator, Senior Chemist at the
“Prof.Dr.I.Chiricuta” Oncology Institute, since 1996
Professional experience in the topic of the project: functional genomics in cancer biology,
molecular medicine • exchange visits for specialization in pharmacology, toxicology and
chemotherapy, • participant in 22 research projects, leader of 2 national/international
projects, principal co-investigator in 3 national projects • 23 ISI-quoted scientific publications;
Publications:
1. Krausz LT, Fischer-Fodor E, et al, GITR- expressing regulatory T-cell subsets are
increased in tumor-positive lymph nodes from advanced breast cancer patients as
compared to tumor-negative lymph nodes. Int J Immunopathol Pharmacol. 2012;
25(1):59-66.
2. Meszaros I, Krausz L. T., Fischer-Fodor E, Expression profiles of immunomodulatory
genes in human solid tumors, Revista Romana de Medicina de Laborator, 2011,19 (1-4):
65-74.
3. Decean H, Perde-Schrepler M, Tatomir C, Fischer-Fodor E, Brie I, Virag P. Modulation of
the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated
human keratinocytes and melanocytes. Arch Dermatol Res. 2013, PMID: 23604927
Affiliation to Professional Societies: •American Chemical Society (ACS) • Romanian Society
of Radiation Therapy and Medical Oncology, ESTRO •Free International Association of
Researchers on Natural Substances 09 -FIARNS 09

Research team members

Brie Ioana Carmen, MD, specialist in radiotherapy, PhD student, ELISA tests for
inflammatory cytokines secreted by lymphocytes from AIED patients in vitro
Soritau Olga, MD, specialist in laboratory medicine, PhD student, treatment of the
separated lymphocyte populations in vitro with inner ear antigens
Barbos Otilia, MD, specialist in dermatology, PhD student, density gradient separation
separation of lymphocytes, and sera from the patients’ blood
Chereches Gabriela, MD, specialist in laboratory medicine, magnetic separation of
lymphocyte populations, in vitro maintaining of lymphocytes
Tatomir Corina, determination of inflammatory cytokines, in the sera of AIED patients
Tulbure Ana, laboratory assistant, will be in charge of receiving of the blood samples from
the clinical settings, recording in the database, storage of samples
Vicovan Marilena, financial administrator
Pop Daniela, aquisitions

Partner 2
Key persons
Udrea Mircea, age 64,

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Career path: 01/09/1967 - 01/07/1972, Polytechnical Institute of Bucharest (University),
diplomate engineer; 01/08/1972 - 01/11/1988, Institute for Atomic Physics (PhD studies),
PhD in Technical Physics
Work experience: 01/08/1972 - 01/09/1999, Senior researcher, National Institute for Laser,
Plasma and Radiation Physics, Magurele
01/03/1999 - 01/08/2001, Professor, Middle East Technical University, Physics Department,
Cankaya, Ankara (Turkey: Laser Design Course, Head of the Laser laboratory, Master and
PhD students; 01/08/2001 - 01/10/2008, Senior Researcher II, National Institute for Laser,
Plasma and Radiation Physics, Bucharest ; 01/10/2008 – 01/06/2012, Senior Researcher I,
National Institute for Laser, Plasma and Radiation Physics; 01/06/2012 – present, General
Manager, Apel Laser srl; Main activity: management, researches on laser applications in
industry and medicine
Competences: High power pulsed gas lasers, diode lasers, laser applications in science,
industry and medicine; pulsed laser deposition systems by using excimer lasers, electrical
measurements and characterizations of materials. In this project he will study the technical
requirements for the realization of a multiplex array for cytokines in AIED
5 OSIM patents in the laser field, 1 International patent WO2010/092301 A1, (2010)
32 papers in ISI quoted journals, over 80 contributions at International Conferences
Chairman of the International Conference INDLAS 2007, 2008, 2013

Chiricuta Bogdan, age: 32

Profession / Current position: Chief of the R&D department, PhD student
Name of employer: APEL LASER SRL
Experience in the project field: Having participated in multiple specific trainings, has an
extensive experience in the field of spectroscopy comprising Raman, fluorescence,
ellipsometry, X-Ray fluorescence, particle size analysis, SPRi and elemental analysis. He will
be in charge with the detection of inner ear antigens in the sera of patients and control
subjects
List of the most significant research projects:
• Research team leader on behalf of SC Apel Laser SRL in the frame of PN-II-PT-PCCA-
2011-3 Sensor systems for secure Operation of Critical Installations - SOCI – project,
contract 8/2012 - coordinator INFLPR Bucharest
• Participant researcher from SC Apel Laser SRL in the frame of the PN-II-PT-PCCA-2011-
3 Process and device for thin films deposition in highly ionized pulsed plasma - PIDESS
project, contract 174/2012 - coordinator "A. I. Cuza" University, Jassy
• Participant researcher from SC Apel Laser SRL in the frame of the PN-II-PT-PCCA-2011-
3 Plasmonic 2D optical memory with active chalcogenide glass layer - MEMOPLAS
project, contract 25/2012 - coordinator Politehnica University, Bucharest
Pending patent: “Laser system adapted for an ophtalmic microscope”

Iacob Liliana Mioara, age 34,

Works at Apel Laser SRL as Physicist, Optics, spectroscopy, plasma and lasers, over 8
years experience in CEEX, Partnerships, and Innovation type research projects which have
resulted in patents, technologies and photonic devices. She will be involved in the realization
of the multiplex array for the detection of cytokines
Participant researcher from Apel Laser SRL in the frame of the PN-II-PT-PCCA-2011-3
Process and device for thin films deposition in highly ionized pulsed plasma– PIDESS
project, contract 174/2012 - coordinator "A. I. Cuza" University Jassy
Responsible on behalf of Apel Laser SRL in the frame of ”Nanostructured oxidic thin films
laser processing for conventional and transparent electronics ” – PROLAF – project
Responsible on behalf of Apel Laser SRL in the frame of “Laser technologies for in situ
covering of orthopedic prostheses with bioactive ceramics” – LASERBIOCER – project
Scientific papers:

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Stănescu S.L., Sava V, Udrea MV, Chiricuta B, Iacob M, Cristea P.D, Adaptable Laser
System for Ophthalmic Microscopes, Romanian Journal of Opthoelectronics, 2011, Volume
16, Issue 1,

Research team members

Sava Vasile, age 28, involved in the market research
Technical Manager at Apel Laser SRL, has over 5 years experience in designing and
building DPSS laser systems.
Rada Andrei-24 years, Msc student will participate in the detection of inner ear antigens by
western blot
Lungu Cristina- 23 years, chemist engineer, biologic sample processing, reagents
preparation
Marin Gheorghe- 56 years, technician
Selagea Mihai Nicolae- 26 years, scientific researcher will be in charge of the statistic
analysis of the results
Vasiliu Manuela Doina, 57 years, financial administrator

3.5. CONSORTIUM COMPLEMENTARITIES AND SYNERGIES BETWEEN PARTNERS

Show the complementarities and added value of the collaboration between partners. The
interdisciplinary and the openness to diverse collaboration must be justified in accordance with
the project orientations.
The multidisciplinarity concept in this project is sustained by the involvement of partners from
different academic areas as well as from the private field.
The professional competence of the participating researchers, having experience in
approaching this kind of multidisciplinary researches, will assure the accomplishment of the
proposed objectives minimizing the risks of failure. The present consortium brings together
physicians from different specialities: ENT, rheumatology, immunology, chemists, biologists,
physicists, engineers, etc. The partners involved in projects, UMFCN (CO) and IOCN (P1)
had previous collaborations in two research grants: CEEX Grant no 183/2006 – Study of the
inner ear sensory epithelia regeneration through development of hair cell cultures in
mammals and CNCSIS Grant no 2620/2008- complex exploratory programs- Protective
effect of the antioxidants against chemotherapeutic agents ototoxicity in the organ cultures of
the rat cochlea, in which significant results were obtained materialized in joint publications in
ISI journals, presentations at scientific meetings. All these previous collaborations, and also
the experience of partners in research grants are making the premises for the future
collaboration very optimistic. The involvement of a new partner, which is Apel Laser SRL a
company which participated in the past in research grants will make possible the
implementation of our research in practice, the results obtained in this project being used to
develop a useful tool for the diagnosis of AIED, a multiple detection kit for the simultaneous
detection of AIED markers.

4. PROJECT MANAGEMENT

For information only: maximum 5 pages for this section without tables and graphs.

The content of this section corresponds to the fourth evaluation criterion (management,
methodology, work plan, milestones and budget).
The management structure and procedures were designed to ensure the following:
• Planning the objectives and monitoring their achievement within budget and schedule.
• Establishing the responsibilities of those involved in the realization of the project.
• Determining the categories of the necessary human and material resources.
• Control of project implementation and the monitoring documents and deliverables quality.
• Communication between partners and between coordinator and Contracting Authority.

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• Establishing the decision-making mechanism.
All partners assume the technical and financial responsibilities for the project
management. The responsibilities in the frame of project management are the following:
Project Coordinator (CO) – through Dr. Alma Maniu ensures the project management
(scientific and financial), technical direction of the project and the operational communication
with the partners and with the Contracting Authority. The project manager will make public the
activities and will announce eventual vacant positions. Also, he drafts and hands in the
intermediary and final reports. The reports will be approved by Consortium Committee before
delivery.
Consortium Committee (CC) - will be composed of one representative member from
each project partners (CO-UMF Dr. Alma Maniu, P1-IOCN Dr. Maria Perde-Schrepler, MD,
P3-Apel Laser). The CC will be consulted by CO on matters of primary importance for
project. The duties of CC are: ►to decide the major general technical direction of the project;
►to take major decisions on project finances and to solve major difference of opinion; ►to
establish plans to reduce the impact of occurring risks. Decisions are taken by voting (2/3 of
votes) within internal meetings convened one month before the reporting period or at the
request of partners, if necessary. Conflicts will be solved at the lowest level possible (WP
level). If it is not possible, the CO will mediate, than the CC will make a decision and if
necessary will ask the Contracting authority for further actions. Procedures will be settled and
signed in the Consortium Agreement before the start of the project.
Work package (WP) leaders deal with the scientific management of the project under
the CO. Their duties are: ►to monitor the progress of the project, to prepare and summit
progress reports and final report indicating the achievement degree of WP in %; ►to ensure
the deliverables on time; ►to participate to project internal meetings. They should warn the
CO about possible difficulties in WP progress and may call for technical meeting if required
for the CC consulting.
The quality of the project management is ensured by the preparation of a Management
Handbook containing administrative forms, templates, reporting periods, etc.
The risks that may affect the progress of the project can arise from unexpected technical
difficulties/scientific findings, poor communication or cooperation within/between partners,
irregular financing. By proper management and good communication among partners,
respecting the protocols and ensuring every member of the project perform their tasks these
risks will be eliminated.

4.1. WORK PLAN, DELIVERABLES AND LOAD BALANCING

Describe “who does what”: present the scientific programme for all partners and justify the work
programme breakdown into work packages (WPs) consistent with the objectives.
Describe the project WPs taking into account the contribution of each partner. For each WP
describe:
- the goals and success indicators if any,
- the WP leader and the partners involved (this will be indicated in the tables below),
- the detailed work programme,
- the deliverables,
- the contributions of the partners (“who does what”),
- a description of the methods and technical choices and the way in which solutions will be
brought,
- the risks and back-up solutions envisaged.

WORK PACKAGE LIST

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 Work
Work Start
packag Person/month End
Work package title package 3 mont
e month5
leader2 h4
No1

WP1 Establishing of the study CO 6.29 1 3

protocols, databases, web
page

WP2 Clinical study CO 23.9 2 24

WP3 Immunologic study P1 23.46 2 22

WP4 Determination of specific P2 36.23 2 22

markers

WP5 Dissemination of the results CO 11.07 4 24

WP6 Data analysis and CO 4.7 18 24

interpretation

TOTAL 105.65

Using the table below, indicate the description for each work package, specifying the technical
and scientific milestones, the bottlenecks or contingencies that could jeopardize the project
outcome, and the planned project meetings.

WORK PACKAGE DESCRIPTION

WP no. 1

WP title Establishing of the study protocols, databases, web page

WP leader CO

Involved
CO P1 P2 Total
partners
Person-months 2,3 2,09 1,9 6,29
Start month 1

1 Work package number: WP 1 – WP n.

2
Number of the partner leading the work in the WP
3
The total number of person-months allocated to each work package.
4
Relative start date for the work in the specific work packages, month 1 marking the start date of the
project, and all other start dates being relative to this start date.
5
Relative end date, month 1 marking the start date of the project, and all end dates being relative to
this start date.

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 End month 3
Objectives
1. Establishing the inclusion and exclusion criteria for the patients to be enrolled in the
study as well as of the control subjects, preparing the forms for the informed consent
2. Establishing experimental protocols
3. Elaborating the databases for the results
4. Construction of the projects’ web page
Description of work and role of participants
WP responsible: Alma Maniu
This work package is of great importance for the success of this project because it establishes
the foundation on which every result will be built. A correct study design will ensure the
obtaining of valuable results with an important scientific impact. CO will coordinate this WP
being involved in the development of examination protocols for the patients to be enrolled in the
study as well as of the control cohort. This will be a collaborative work between the physicians
from the E.N.T. department and rheumatology department.
Objective 1. CO- responsible: Alma Maniu; participants: Violeta Necula, Laura Damian
Therefore two groups of patients will be set:
The first group will be made by outpatients suffering from sensorineural hearing loss (coming
first to outpatient E.N.T. hospital) and the second group will be made by outpatients suffering
from ANCA positive vascullitis addressing first to the rheumatology outpatient hospital. The
examination protocol will consist of: 1. Medical history of the crisis; 2. Clinical E.N.T.
examination (nasal endoscopy, endoscopic examination of oropharynx and larynx , otoscopy,
otomicroscopy, tuning fork tests, pure-tone threshold evaluation (including AC and BC for 500,
1000, 3000, 4000, 8000 Hz, speech discrimination, tympanogram and acoustic reflex,
otoacoustic emissions, vestibular exam; checking the spontaneous and evoked nystagmus;
head Shaking test; Halmagyi test; Romberg test; Unterberger test; indication test; nose finger
test; VideoFrenzel examination with light and in darkness. 3. Rheumatologycal examination on
each individual case will assess, the presence of signs for autoimmune diseases including
vasculitis, according to BVAS3 examination protocol (developed by Luqmani et al), with clinical
examination focused on disease activity in several systems: general, cutaneous,mucous
membranes/eyes, chest, cardiovascular, abdominal, renal, nervous sysyem. The BVAS score
will be recorded. 4. Blood tests: hemoglobin (Hb), hematocrit (Hct), blood count: erythrocytes
(RBC), leukocites (WBC), platelets, neutrophil granulocytes, lymphocytes, monocytes,
reticulocytes, eosinophil granulocytes, erythrocyte sedimentation rate (ESR), glycemia, urea,
creatinine, liver function tests : transaminases – AST, ALT, triglycerides, total cholesterol, HDL
cholesterol, LDL cholesterol, immunograma: IgA, IgG, IgM, C-reactive protein, anti-neutrophil
cytoplasmatic antibodies (ANCAs), ANCA p, ANCA c, antinuclear antibodies (ANAs),
rheumatoid factor (RF), complement fractions C3 and C4, anticardiolipin antibodies, lupus
anticoagulant, acetylcholine receptor antibody, angiotensin converting enzyme, hepatitis B
viruses, hepatitis C viruses, HIV tests. 5. Chest radiography; 6. Magnetic resonance image with
contrast agents. Following the examination protocol the inclusion and exclusion criteria for the
patients to be enrolled in the study will be: Inclusion criteria: uni or bilateral sensorineural
hearing loss with more than 30 dB lost on 2 minimum frequencies; sudden or progressive
onset; good general health; MRI imaging with no pathological findings. Exclusion criteria:
age; conductive or mixt hearing loss; middle ear disease; history of barotraumas, head trauma,
noise induced hearing loss, meningitis; positive MRI for vestibular schwannoma; positive FTA
(syphilis); positive blood test for Lyme disease; positive tuberculosis test; past treatment or use
of medication or substances to cause ototoxicity (aminoglycosides, cisplatin, loop diuretics);
positive test for HIV; positive for Hepatitis B or C; presence of demyelinating disease, such as
multiple sclerosis; genetic context (other family members with sensorineural hearing loss).
The control group: will be composed from the same number of healthy outpatients; Inclusion
criteria: Outpatients coming first to E.N.T. outpatient hospital. Exclusion criteria: Patients
suffering from inflammatory E.N.T. pathology or malignancies of the ear nose and throat.

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Preparing the forms for the informed consent of the subjects
The investigators represented by the doctors from E.N.T. department and rheumatology
department will fully inform the subject about the study. Before informed consent may be
obtained, the investigator, or a person designated by the investigator, will provide the subject or
the subjects’ legally accepted representative ample time and opportunity to inquire about
details of the trial and to decide whether or not to participate in the trial. All questions about the
trial should be answered to the satisfaction of the subject or the subjects’ legally accepted
representative. The written informed consent form will include explanations of the following:
that the trial involves research; the purpose of the trial; the trial treatment(s) and the probability
for random assignment to each treatment; the trial procedures to be followed, including all
invasive procedures; the subjects responsibilities; the reasonably expected benefits; the
alternative procedure(s) or course(s) of treatment that may be available to the subject, and their
important potential benefits and risks; the compensation and/or treatment available to the
subject in the event of trial-related injury; the expected duration of the subject’s participation in
the trial.
Also the approval of the Ethical Committee of “Iuliu Hatieganu” University of Medicine and
Pharmacy will be requested for the study.
Objective 2. P1- responsible: Maria Perde-Schrepler, will elaborate the protocols for the in vitro
study of the immunologic profile of the patients compared with healthy subjects.
P2- responsible: Becherescu Barbu Dan Nicolae will establish the protocols and methods to be
used for the determination of the inner ear antigens.
Objective 3. responsible: CO: Oana Harabagiu PhD student,
The databases for the results will be elaborated during this first activity. The study will be
prospective transversal. The databases will comprise selected patients from the patients
addressing to E.N.T. and rheumatologycal regional ambulatory Cluj-Napoca. The database will
be stocked in a special created program for statistical analysis.
Objective 4. CO- responsible: Alma Maniu; participant- postdoc Cătana Iuliu Vlad, will define
the purpose of the projects’ Website; will gather materials and information for the website;
elaborate the writing text; review and send the website materials;
Risks and Backup solutions:
• Small number of subjects, non-homogenous cohorts could lead to inconclusive results.
To ensure a sufficient number of subjects included in the trial, patients will be actively
recruited, by collaborating with specialists from other units and family doctors; in order
to obtain homogenous cohorts, the inclusion and exclusion criteria will be elaborated
with great care by experimented physicians and will be strictly followed. The patients will
be stimulated to enter the study by elaborating an informed consent which will highlight
the benefits of their participation.
• To avoid unreliable results in the experimental study the protocols will be elaborated by
using the most modern, sensitive and high quality assays coming from reliable R&D
suppliers.
• Not obtaining statistical significance- besides having a sufficient number of subjects
included in the study, databases will be elaborated in order to apply the best statistic
methods; Experimented statisticians will collaborate to obtain the best results
Deliverables (brief description and month of delivery)
• Clinical and paraclinical algorithm of the study- includes all the clinical and paraclinical
methods used in the diagnostic of each patient as well as the treatment schedules;
(month 3)
• Experimental protocols for the evaluation of the immunologic status and lymphocytes
populations- laboratory protocols (month 3)
• Experimental protocol for the determination of inner ear markers (month 3)
• Web page (month 3)
• Database, patients registries (month 3)
WORK PACKAGE DESCRIPTION

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 WP no. 2

WP title Clinical study

WP leader CO

Involved
CO Total
partners
Person-months 23,9 23,9
Start month 2
End month 23
Objectives
1. Enrolment of patients and healthy subjects
2. Clinical diagnosis, based on clinical exam and history of the disease
3. Paraclinical examinations, lab tests
4. Initiation of treatment
5. Follow-up, evaluation of response to treatment: repeated audiograms, patients
subjective improvement
Description of work and role of participants
WP responsible: Alma Maniu
Objective 1. Enrolment of patients and healthy subjects- WP responsible: Violeta Necula
participants: Maria Petri, Linda Ghib
In this activity the patients and healthy subjects will be enrolled according to the inclusion and
exclusion criteria and they will sign the informed consent. All patients will be hospitalized for
one day and a detailed history of the disease will be performed. The same number of control
group patients will be enrolled.
Objecxtive 2. Clinical diagnosis- responsible: Alma Maniu ; participants: Laura Damian, Violeta
Necula, PhD student Maria Petri
CO team will perform a complete E.N.T clinical exam on patients At this point the patient circuit
throughout the hospital will be the follow: Audiological examination performed in the ENT Clinic
Audiology Department of the Clinical County Emergency Hospital. The audiological protocol will
include the pure-tone threshold for air and bone conduction for the hearing impairment
detection, speech perception and discrimination for social assessment, tympanogram and
acoustic reflex for middle ear analysis and otoacoustic emission (transitory and distortion
products) which are important for inner ear function assessment (they represent responses of
the cochlear CCE. The vestibular examination will be performed in the following way: Medical
history of the crisis, the most important component in the diagnostic of the dizzy patient and
include a classification of the symptoms, duration, triggers and associated symptoms; Checking
of the spontaneous nystagmus and the gaze evoked nystagmus. Head Shaking test as a part
of oculomotor examination in assessed for vestibular disfunction; Halmagyi test detects severe
unilateral function loss of the vestibular system and can help in making a distinction between a
cerebellar infarction and a vestibular neuritis; Romberg test is used in checking the body's
sense of positioning (proprioception) A loss of balance is interpreted as a positive Romberg's
test; Unterberger test for helping assess whether a patient has a vestibular pathology; Nose
finger test usefull for detecting the cerebellar disorders so called dysmetria which refers to a
lack of coordination of movement typified by the undershoot or overshoot of intended position;
Positional manoeuvers that have the purpose to try to elicit vertigo and nystagmus;
VideoFrenzel examination with light and in darkness which offers a better view of the eye
movements and provides a better diagnostics of the dizzy patients
3.Lab tests, chest X-ray, MRI- responsible: Laura Damian, participants: Mihaela Popescu,
Gabriela Ciursa.

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A certain amount of blood will be taken from the patients by the chief nurse. Blood will be also
harvested for the bank for serum separation and preservation for the serum bank at the first
visit, after 3 months, after 6 and 12 months respectively. The other medical doctors will make
the appointments for the chest X-ray and MRI. Patients will undergo a complete
rheumatologycal examination within the rheumatology department, according to the afore-
mentioned procedure. The clinical diagnosis will be established. All data will be stocked in the
database. Final diagnosis will be set at the second visit, after a complete evaluation of all
results.
4. Initiation of treatment: responsible: Alma Maniu; participants: Violeta Necula, Laura Damian.
Patients will be divided into two groups according to diagnosis: patients suffering from AIED
and patients with AIED and associated vasculitis. The treatment will be initiated with high doses
of oral steroids. Hypertensive and diabetes patients will be injected with intratympanic steroids.
Vasculitis patients will ne treated with cyclophosphamide and methylprednisolone for remission
induction, followed by azathioprine and/or methotrexate for remission maintenance according to
the EUVAS protocol.
5. Follow-up: responsabil: Oana Harabagiu- PhD student; participants: postdoc Iuliu Vlad
Catana, Linda Ghib
All patients will be equally monitored for treatment response by performing control audiograms
at 1, 3 and 6 mounth. Evaluation of response to treatment: repeated audiograms, patients at 1,
3. 6 months
Audiological follow-up will consist of pure-tone threshold detection, speech discrimination tests,
otoacoustic emissions, tympanogram and acoustic reflex and also the patients, subjective
improvement of hearing.
Risks and Backup solutions:
• inclusion of an insufficient number of subjects in the study for the statistic significance of
the results; this will be eliminated by an intensive recruiting at both clinical sites,
collaborations with other clinics and physicians, collaborations with family doctors in
order to increase awareness to identify potential subjects and orient them towards the
recruiting centres; The clinical sites involved in this project benefit of a great
addressability providing care for patients from the entire region of Transylvania.
• refusal of patients to be included in the study will be avoided by the elaboration of the
informed consent which will inform the patient of all the benefits of participating in such
a study;
• adverse effects of therapy- in this respect, patients will be tested at the beginning of the
trial, and treatments will be done according to each patients’ individual medical status.
• patients’ withdrawal from the study either voluntarily or due to other causes (additional
illness, moving, etc.); this inconvenient will be minimized by an active follow-up, by
keeping track of patients addresses, phone numbers, reminding them of their
appointments.

Deliverables (brief description and month of delivery)

• Bulletins recording clinical symptoms and MRI at the beginning of the study (month 18)
• Bulletins of laboratory analyses at enrolment (m.18)
• Bulletins of repeated audiograms for each patient (m. 23)
• Patients registry with the evolution of patients from enrolment and follow-up (m.23)
WORK PACKAGE DESCRIPTION

WP no. 3

WP title Immunologic study

WP leader P1

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 Involved
P1 Total
partners
Person-months 23.46 23.46
Start month 2
End month 22
Objectives
1. Receipt of blood samples from the clinical settings, separation of sera, separation of
lymphocytes, storage (-80ºC freezer)
2. Determination of inflammatory cytokines
3. Determination of lymphocytes populations
4. Evaluation of the effects of inner ear antigens on the lymphocytes populations of
patients with AIED compared to healthy subjects
Description of work and role of participants
WP responsible: Maria Perde-Schrepler
This WP is based on the hypothesis that patients with AIED have a systemic immunologic
disease manifested in the inner ear being initiated by a cochlear antigen. The research team of
Partner 1 will investigate the behaviour of the immunocompetent cells in patients with AIED
compared to healthy subjects. Also the presence of inflammatory cytokines in the sera of the
subjects (both patients and controls) will be evaluated. The existence of a specific immunologic
pattern will be pursued and this could be included in the future diagnostic protocol for this
disease.
Objective 1. P1- responsible: Gabriela Chereches, participants: Otilia Barbos PhD student,
Ana Tulbure.
The biological samples will be received from the clinical settings, registered and processed for
the future analyses. One tube (venous blood on EDTA) will be centrifuged for the separation of
serum and stored at -80°C; the other one, on hepari n will be centrifuged in gradient density (on
Ficol) for the isolation of mononuclear cells. The separated cells will be resuspended in freezing
medium and stored at -80°C, then at -180°C until f urther processing.
Objective 2. P2- responsible Piroska Virag, participants: Ioana Brie PhD student, Corina
Tatomir. The serum of AIED patients and control subjects will be analysed regarding the
cytokine content. The following cytokines will be assessed: IL1α, IL1β, IL2, IFNγ, TNFα. The
method to evaluate cytokine levels will be ELISA using dedicated kits.
Objective 3. P2- responsible: Eva Fischer-Fodor, participants: Olga Soritau PhD student,
Maria Perde-Schrepler. The mononuclear cells, separated by gradient density centrifugation
will be thawed and resuspended in culture medium for lymphocytes. They will be cultured in
CO2 water jacketed incubators at 37°C. One part of the lymphocytes will be further separated
by immunomagnetic cell sorting, using a MidiMACS unit. The lymphocytes will be treated with
CD4 and CD8 marked magnetic beads. The obtained lymphocyte populations will be counted
and cultivated in specific media for further experiments.
Objective 4. P2- responsible: Maria Perde-Schrepler; participants, Piroska Virag, Oga Soritau.
The sorted lymphocytes will be cultivated and exposed to inner ear antigen (recombinant
cochlin). For comparison, untreated (negative control) lymphocytes and lymphocytes treated
with lypopolisaccharid (LPZ) (pozitive control), LPZ being a known inflammation inducing agent.
The response of the treated lymphocytes will be evaluated by: cell proliferation and the
secreted cytokines, evaluated before and after treatment. Three categories will be tested:
patients with AIED who responded to treatment with corticosteroids; nonresponders; control
subjects.
Risks and Backup solutions:
• Losing of samples due to improper handling, storage, infections will be avoided by
conducting the experiments according to the good laboratory practice principles: the
samples will be transported immediately after prelevation, and stored adequately.
Separations will be performed in perfect sterile conditions, under laminary flow hoods,

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 samples will be registered and stored.
• All reagents will be ordered from the same batches and from reliable suppliers to avoid
interexperimental differences;
• Temporary malfunctions of the research equipment will be eliminated by existing
backup devices in the research infrastructure of the team.
Deliverables (brief description and month of delivery)
• Serum sample bank (m. 18)
• Results bulletins concerning the cytokines involved in AIED (m.22)
• Results bulletins of the lymphocytes populations profiles (m.22)
• Results bulletins with the modulations of the lymphocytes populations by inner ear
antigens (m.22)

WORK PACKAGE DESCRIPTION

WP no. 4

WP title Determination of specific inner ear antigens

WP leader P2

Involved
P2 Total
partners
Person-months 36,23 36,23
Start month 2
End month 23
Objectives
1. Receipt of sera from the clinical settings
2. Determination of circulating inner ear antigens and antibodies from the sera of patients
and control subjects
3. Realization of an experimental multiplex array assay for the simultaneous quantitation of
the specific cytokines found increased in the serum of AIED patients.
4. Market research, for the evaluation of the marketable potential of such a multiplex kit
Description of work and role of participants
WP responsible: Nicolae Dan Becherescu Barbu
This WP is based on the hypothesis that as a result of the fact that inner ear antigens are
recognized as non-self by the immune system, circulating inner ear antigens as well as
antibodies against them could be quantified in the serum of patients with AIED.
Objective 1: Responsible: Mioara Iacob; participants: Mihai Nicolae Selegea, Gheorghe Marin.
When a significant amount of sera from patients and healthy subjects are collected at the site of
Partner 1, a transport will be organized to the site of Partner 2, the samples being transported
on dry ice. At the site of Partner 2, the samples will be recorded and stored until further
processing.
Objective 2: Responsible: Bogdan Chiricuta, participants: Cristina Lungu, Vasile Sava, Andrei
Rada, Mioara Iacob, Nicolae Becherescu
The serum of patients will be analyzed regarding its content in antibodies against cochlin, β-
tectorin, collagen II and IV, using ELISA kits. Heat Shock Protein (HSP70) will also be
assessed by Western Blot. All patients will be evaluated in parallel with the healthy subjects.
Objective 3: Responsible: Virgil Udrea; Participants: Vasile Sava, Bogdan Chiricuta, Manuela
Vasiliu, Cristina Lungu, Gheorghe Marin

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After the validation by the existing correlations with the clinical evolution of the patients, the
selected markers which proved statistic significance for the diagnosis of AIED will be integrated
in a multiplex detection kit. The development of multiplex immunoassays requires rigorous
validation of assay configuration and analytical performance to minimize measurement
imprecision. Optimization of capture ligand selection and immobilization, assay calibration,
assay performance (determination of incubation times, reduction of cross-reactivity between
assays, non-specific protein binding) will be performed. The accuracy of quantitative
immunoassays depends on the development and validation of appropriate calibrators and
calibration methods. For the elimination of assay interference between reagents, cross
reactivity between reagents will be evaluated. Antibody-related assay interference will be
evaluated by 3 experiments: 1) single proteins incubated with complete detection antibody
cocktail; 2) complete protein mixture incubated with single antibodies; 3) antibody cocktail with
one antibody removed and incubated with complete protein mixtures. Optimal temperatures,
incubation times and concentrations of reagents will be configured by using a multifactorial
design. Analytical validation will include: range of linearity, analytical specificity, recovery, limits
of detection and quantification, reasonable imprecision, and comparison to a quality reference
method.
Objective 4: Responsible: Nicolae Becherescu, Participants: Vasile Sava, Mihai Selegea,
Mioara Iacob, Gheorghe Marin
This activity is of great importance in deciding whether further patenting and research costs are
justified. The following issues will be addressed: key features and the benefit of the technology;
the competences of the organization in this technology area; identification of the further
beneficiaries of the technology; comparison with the competitors; next steps and further work
required.
Risks and Backup solutions: the deterioration of the samples during transport to the site of
partner 2 (a 7-8 hour drive) will be prevented by transporting the serum samples in containers
on dry ice, thus avoiding the thawing of the samples. After reception, registration, the samples
will be stored in -20°C freezers, until further pro cessing. All reagents, kits which will be used will
be ordered from reliable providers to ensure the experiments’ reproductibility. The planned
development of the multiplex assay could be cost- ineffective, and in this respect, a complex
feasability study and market research will be conducted.
Deliverables (brief description and month of delivery)
• Results bulletins regarding the inner ear antigens (m.18)
• Multiplex array for AIED markers- in the phase of development/ validation (m.23)
• Market research report (m.23)

WORK PACKAGE DESCRIPTION

WP no. 5

WP title Dissemination of the results

WP leader CO

Involved
CO P1 P2 Total
partners
Person-months 5,4 4.47 1.2 11,07
Start month 4
End month 24

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Objectives
1. Presentations of the results at conferences, work-shops, publications
2. Round tables
Description of work and role of participants
Dissemination of the results will be supervised by the CO.
The persons in charge with this WP will be the research team leaders of each partner. All
partners will be involved in the dissemination of the projects’ results by participating to national
and international scientific meetings and conferences. Also the results obtained during the
project will be published in ISI journals. The web page of the project will be updated to inform
the public about the achievements of the project.
At the end of each year a round table will be held with the participation of all partners and
invited specialists from the field. The obtained results will be presented, all the issues and
eventual difficulties will be discussed between specialists with high expertise, in order to
improve the quality of the research. The CO will be involved in organizing a round table on this
subject. During this round table ample discussions will take place. The oncological department
will present their results regarding inflammatory cytokines, lymphocytes populations and the
effects of inner ear antigens on the lymphocytes populations of patients with AIED compared to
healthy subjects. The aspects obtained regarding the presence of inner ear antigens in the
patients’ serum and their involvement in the ethiopathogenesis of the disease will be
underlined. The consortium will validate the diagnostic and treatment protocols in autoimmune
inner ear disease based on objective data resulted from detailed statistical analysis.
Deliverables (brief description and month of delivery)
• Presentations published in abstract books (m. 5-24)
• Publications in ISI journals (m. 8-24)
• Round table (m.12 and m.24)

WORK PACKAGE DESCRIPTION

WP no. 6

WP title Data analysis and interpretation

WP leader CO

Involved
CO P1 P2 Total
partners
Person-months 1.60 1.60 1.50 4,7
Start month 18
End month 24
Objectives
1. Statistic analysis, establishing correlations of response to treatment with the laboratory
findings
2. Elaboration of final protocols of diagnosis and treatment based on the obtained results
Description of work and role of participants
Objective 1. All partners: CO- responsible: Alma Maniu; P1- responsible Maria Perde-
Schrepler; P2: Becherescu Barbu Dan Nicolae will analyze the results obtained by their
research team. Statistic analysis will be performed, establishing correlations of the patients’
response to treatment with the serologycal markers behavior. Patients will be categorized as

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steroid responders and steroid nonresponders. Statistical correlations will be in order to obtain
new answers regarding the complex physiopathogenetic mechanisms involved in the studied
disease.
Objective 2. Based on these results, the project coordinator (CO)- responsible Alma Maniu,
together with the partners will elaborate new protocols for the diagnosis and treatment of AIED,
of which will benefit all the medical specialties which have to deal with this disease and most
important the patients will be faster diagnosed and better treated.
Risks and Backup solutions
Insufficient data, incorrect results, lack of correlations- the solution for these problems is a
correct study design, the enrollment of a sufficient number of subjects, the reporting of results
in time as stated in the agreement between partners.
Deliverables (brief description and month of delivery)
• Completed databases and registries (m.23)
• Validated protocols for the diagnosis and treatment of AIED (m.24)

A table summarizing all the project deliverables will be provided below.

LIST OF DELIVERABLES

Nature of Disseminatio
Del. WP WP Delivery
Deliverable name deliverable n
no. 6 no. leader 7 date9
level 8
Clinical and
1 CO FM CO month 3
paraclinical protocol
1 to be performed on
each patient
Experimental
1 CO EM CO month 3
protocols for the
evaluation of the
2 immunologic status
and lymphocytes
populations
Experimental
1 CO EM CO month 3
protocol for the
3 determination of
inner ear markers
Web page
4 1 CO EM PU month 3
Database, registries
5. 1 CO EM CO month 3
Bulletins recording
2 CO FM CO month 18
6 clinical and
paraclinical

6 Deliverable numbers in order of delivery dates: D1 – Dn

7
Please indicate the nature of the deliverable using one of the following codes:
EM = Experimental Model; FM= Functional Model; P = Prototype, D = Demonstrator/ Demonstrative
model, IT = Innovative Technology, IS = Innovative Services.
8
Please indicate the dissemination level using one of the following codes:
PU = Public
PP = Restricted to other programme participants (including the Contracting Authority)
RE = Restricted to a group specified by the consortium (including the Contracting Authority)
CO = Confidential, only for members of the consortium (including the Contracting Authority)
9
Month in which the deliverables will be available. Month 1 marking the start date of the project, and
all delivery dates being relative to this start date.

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 evaluations at the
beginning of the
study
Bulletins of
2 CO FM CO m.18
7 laboratory analyses
at enrolment
History of repeated
2 CO FM CO m. 22
audiograms for each
8 patient

Patients registry with

2 CO FM CO m. 23
9 the evolution of
patients
Serum sample bank
10 3 P1 IS CO m. 18
Results bulletins
3 P1 EM CO m.22
concerning the
11 cytokines involved in
AIED
Results bulletins of
3 P1 EM CO m.22
the lymphocytes
12 populations profiles

Results bulletins
3 P1 EM CO m.22
with the modulations
13 of the lymphocytes
populations by inner
ear antigens
Results bulletins
4 P2 EM CO m.22
regarding the
presence in the sera
14 of patients of
specific inner ear
markers
Experimental
4 P2 IT CO m.23
15 multiplex array for
AIED markers
Market study report
16. 4 P2 FM CO m.23
Presentations
5 CO EM PU m. 5-24
published in abstract
17. books

Round table
5 CO EM PU m.13 and
18. program
m.23
Publications in ISI
19. 5 CO EM PU m 8-24
journals
Completed
6 CO FM CO m.23
databases and
20. registries

Validated protocols
6 CO FM PU m.24
21. for the diagnosis and
treatment of AIED

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4.2. COORDINATION AND TASK SCHEDULE

Present the task schedule and the corresponding sequencing using a Gantt chart.
2013 2014 2015
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
WP
1
1
2
3
4
WP
2
1
2
3
4
5
WP
3
1
2
3
4
WP
4
1
2
3
4.
WP
5
1
2
WP
6

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1
2

Legend CO P1 P2 All partners

4.3. AVAILABLE RESEARCH INFRASTRUCTURE, AND ITS UPGRADE/DEVELOPMENT

List the main research equipment (with a brief description) and infrastructure available at
each partner, related to the project topic and specific tasks.
Specify if and how the infrastructure will be upgraded and/or developed during the project
lifetime.
Research infrastructure
CO- UMFCN
◄Equinox 2.0 Audiometer - full battery of audiometric tests, Otoaccess database software.
◄nteracoustics video Frenzel VF 405; for superior nystagmus observation, provides ideal
conditions for fixation-free observation of eye movements during spontaneous testing,
positional and Dix-Hallpike testing, Head Impulse testing, ◄The Intelligent Hearing Smart EP
Systems; ◄P300/MMN software module eABR software module and hardware to
determinate the Vestibular Evoked Myogenic Potential (VEMP) (the function of the otolithic
organs- utricle and saccule) of the inner ear; ◄Hortman tympanometer Ca 540 ; GSI Auto
VSI- to assess the condition of the ear drum and middle ear. ◄Serena Microscope from Carl
Zeiss - SENSERA model SIP-NO-6628505231. Superlux® 180 W Xenon light source, focus
200-500mm, 12,5 magnetic widefield eyepiece180° til table tube, focal length f=170mm
◄Flow cytometer FacsCantoII (BD Biosciences)- capable of simultaneously collecting data
from up to six parameters simultaneously. ◄A complete proteomic line for protein analysis
for AB SCIEX, as well as a recombinant DNA unit.
P1- IOCN
◄ Laminar flow hoods for the sterile manipulations of biological samples, lymphocytes in
culture◄ Heto-Holten, Revco and Sheldon CO2 incubators- for the in vitro culture of
lymphocytes◄ Hettich centrifuges with cooling system- used for the separation of sera and
lymphocytes ◄ Heto Ultra Freeze freezer –800C- for the storage of frozen samples and for
the serum sample bank ◄Olympus inverted phase microscope- for the live observation of
cells in culture ◄ Tecan Sunrise Elisa reader with washer – for the determination of
cytokines◄BioTek Synergy 2 microplate reader- for the evaluation of several cellular
parameters in absorbance or fluorescence◄ Heidolph Titramax agitator with incubator ◄
Mini Protean vertical electrophoresis device- for the electrophoresis of proteins in gels for
western blot ◄ UV-VIS Spectrophotometer ◄Liquid nitrogen tanks- for storage and transport
of biologic samples and cells◄ Analytical balance ◄ Agilent 2100 Bioanalyzer ◄
Quantitative PCR (Roche) ◄ Full analyses system for automated sequencing and control of
sequence evaluation and fragments in gel- for the evaluation of Western Blot gels ◄ Epics
profile flow cytometer
APEL LASER SRL
◄ RF excited CO2 lasers, output power(s) [40 – 250 W]; ◄ Excimer laser, 193 nm output,
pulse width 10 ns, 10mJ/pulse; ◄Fiber optic coupled laser diode, 808nm/1 W; ◄Laser
diodes: emission @ 408 nm, 635 nm, 650 nm, 780 nm, 808 nm,970 nm; ◄DPSS (diode
pumped solid state laser) 1060 nm; ◄DPSS frequency doubled, 532 nm, 1 W (produced by
Apel Laser); ◄Laser engraving system with optogalvanic scanner (integrated by Apel Laser);
◄Laser cutting / engraving CNC system, 600 X 500 mm; ◄Laser measurement and control
systems (energymeters, powermeters, optical sensors) ◄Spectrometer; ◄Optical table and
opto-mechanical components (mounts, supports, lenses, laser mirrors); ◄Electronic
equipment (multimeters, Tektronix oscilloscope); ◄Chillers for high power lasers (developed

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and produced by Apel Laser); ◄Low level laser therapy systems (developed and produced
by Apel Laser)

During the project the infrastructure will be upgraded and developed by the acquisition of the
following devices:
Equipment name and Justification
characteristics
Coordinator Zeiss microscope Permits a precise examination of patients’ middle
(CO) ear combined with the possibility of surgical
treatment in specific cases.
Storz Endoscopic Light source and ear rigid endoscope used for
equipment ENT screening of all patients with ANCA-
associated vasculitis.
Statistical Program Data processing (patients database during the
study with all the serologic tests and audiological
measurements).
CAM Video System Permits the recording of images from the ear in
all the patients during the diagnostic examination.
Digital Photo Camera Rheumatological screening for all patients
diagnosed with AIED in order to identify signs of
systemic disease.
Partner 1 Up-grade for Zeiss Permits experiments regarding cell culture
microscope: stage-top dynamic and evolution in time, allowing the
incubator with maintaining of viability for the cell cultures,
temperature, humidity and lymphocytes being highly sensitive to
CO2 adjustment system temperature, humidity and CO2 variations
Automated cell counter Offers easy and accurate cell counts and
average cell size, offering reliable data regarding
the number of different lymphocytes populations.
Automatic plate washer Automatic, precise system for optimized washing
of cells, beads and ELISAs
Computers Data processing and image analysis
Laboratory washing Allows cleaning of large volume of glassware and
machine containers. The mechanical cleaning allows the
combination of washing, neutralization and
rinsing cycles, ensuring high quality results
Bench-top centrifuge with For separation of lymphocytes in gradient
high-performance, up to density, separation and purification of serum,
25000xg, refrigeration purification of proteins by ultrafiltration, etc.
Computers Data processing, image analysis
Partner 2 Contact spotting system For the positioning of markers
Computers, Printers Data processing, drafting
Freezer Samples preservation

4.4. MANPOWER ALLOCATION

Using the table below, specify the allocation of person-months for the research team
members, related to the complexity of their tasks.

LIST OF RESEARCH TEAM MEMBERS

First name and Position in the Person-month** Personnel cost

last name* project (Euro)

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 Maniu Alma Project director, 0.3499 31,884.53
lecturer, MD, PhD
Necula Violeta Key person, 0.1768 16,114.92
lecturer, MD, PhD
Damian Laura Key person, MD, 0.1768 16,114.92
PhD,
Harabagiu Oana member, MD, PhD 0.2029 12,201.30
Elena student
Catana Iuliu Vlad member- MD, 0.1340 8,057.46
Coordinator PostDoc
(CO) Petri Maria member- MD, PhD 0.0593 3,568.30
student
Ghib Linda member- MD, CS 0.0938 5,640.22
Popescu Mihaela member- nurse- TS 0.0459 690,64
Ciursa Gabriela member- nurse- TS 0.0891 690,64
Nistor Mariana member - financial 0.0297 230.21
administrator- TS
Raducu member – 0.1485 4,604.26
Loredana Diana acquisitions- TS
Perde-Schrepler Research team 24,172
Maria leader, MD, CS 0.2653
Virag Piroska key person, 11,740
biologist, PhD, CS
III 0.1288
Eva Fischer- key person, 12,661
Fodor Chemist, PhD, CS
III 0.1389
Brie Ioana Member, MD, CS 5,755
0.0999
Olga Soritau member, MD, PhD- 5,755
student 0.0999
Partner 1 Barbos Otilia member, MD, PhD- 6,215
student 0.1078
Chereches member, MD, CS 6,215
Gabriela 0.1078
Tatomir Corina member- Chemist, 6,215
CS 0.1078
Tulbure Ana member- 2,992
technician, TS 0.1007
Vicovan Marilena member- financial 3,453
administrator, TS 0.1114
Pop Daniela Member- 2,302
aquisitions, TS 0.1782
Becherescu Research team 0.2058 18,757
Partner 2 Barbu Dan leader, CS,
Nicolae engineer
Udrea Virgil key person / 0.1176 10,718
Mircea physicist, PhD
Sava Vasile member / CS, PhD 0.1764 16,078
Chiricuta Bogdan Key person/ 0.1176 10,718
physicist, PhD
student

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 Iacob Mioara member/ physicist, 0.1176 10,718
PhD
Rada Andrei member/ Msc 0.1176 7,072
student
Lungu Cristina member/ chemist 0.1176 10,718
engineer, CS
Marin Gheorghe member/ 0.3529 10,939
technician, TS
Selagea Mihai Member/ CS 0.1764 10,608
Nicolae
Vasiliu Manuela member- financial 0.2014 6,243
Doina administrator, TS
105.6513 299,852.66
Total
* Indicate also the non-permanent staff (vacant positions) to hire – doctoral and post-doctoral
researchers, fixed-term contract staff, interns, etc.
** To be indicated with respect to the total project duration

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4.5. PROJECT BUDGET AND PARTNERS SHARE

Present the overall project budget, according to the eligible costs structure, and budget
breakdown by category of expenses.
Specify the budget split among partners from both funding sources (public budget and own
budget).

Budget breakdown / destination (EURO)

Personnel Indirect
Logistics* Travel Total
costs costs

Public 99,797.41 59,940.60 16,575.34 19,367.83 195,681.

Coordi Budget 20
nator
(CO) Own 0 0 0 0 0
Budget
Public 87,481.01 75,007.59 9,208.52 12,473.41 184,170.
Partner Budget 54
1 Own 0 0 0 0 0
Budget
Public 103,365.71 53,179.24 0 39,136.24 195,681.
Partner Budget 20
2 Own 9,208.52 63,999.26 4,834.47 8,287.67 86,329.9
Budget 4
299,852.66 252,126.69 30,618.33 79,265.15 661,862.
Total
88
* Subcontracting cost will be no more than 15% of the project public budget.
Mention/detail subcontracting cost here.

SUBCONTRACTING:
Total: 4671.79 Euro

CO- 3074.56 Euro- 1000 for external audit and 3074.56 for the realization of the web page
P1- 938.4 Euro- for external audit
P2- 658.83 Euro – for external audit

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5. References

Include the list of bibliographic references used in the “State of the art" section and the partners'
bibliographic references related to the project.

References

1. Ruckenstein MJ, Autoimmune inner ear disease, Curr.Opin.Otolaryngol.Head Neck

Surg., 2004, 12: 426-430
2. McCabe BF, Autoimmune sensorineural hearing loss. Ann Otol Rhinol Laryngol.
1979;88:585-589
3. Mijovic T, Zeitouni A, Colmegna I, Autoimmune sensorineural hearing loss: the
otology rheumatology interface, Rheumatology, 2013, 52: 780-789
4. Orozco CR, Niparko JK., Richardson BC., Dolan DF., Ptok MU, Altschuler RA.,
Experimental model of immune-mediated hearing loss using cross-species
immunization, The Laryngoscope, 100 (9): 941-947
5. Hughes GB, Kinney SE, Barna BP, Calabrese LH., Practical versus theoretical
management of autoimmune inner ear disease. Laryngoscope. 1984 ,94(6):758-67.
6. Trume DR, Kempton JB, Mitchell CR,Hefeneider SH, Failure of elevated heat shock
protein 70 antibodies to alter cochlear function in mice, Hear Res.,1998, 116: 65-70
7. Tebo AE, Szankasi P., Hillman TA, Litwin CM, Hill HR, Antibody reactivity to heat
shock protein 70 and inner ear-specific proteins in patients with idiopathic
sensorineural hearing loss, Clinical and Experimental Immunology, 2006, 146: 427–
432
8. Ikezono T, Omori A, Ichinose S, Pawankar R, Watanabe A, Yagi T, Identification of
the protein product of the Coch gene (hereditary deafness gene) as a major
component of bovine inner ear protein, Biochim.Biophys.Acta, 2001, 1535: 258-265
9. Grabski R, Szul T, Sakasi T, Timpl R, Mayne R, Hicks B, Sztul E, Mutations in COCH
that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix
deposition of cochlin, Hum.Genet. 2003, 113:406-416
10. Nair T, Kozma KE, Hoefling NL, Kommareddi PK, Ueda Y, Gong TW, Lomax MI,
Lansford CD, Teliam SA, Satar B, et al, Identification and characterization of choline
transporter-like protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target
of antibody- induced hearing loss, J.Neurosci., 2004, 24: 1772-1779
11. Billings P, Experimental autoimmune hearing loss, J.Clin.Invest., 2004, 113: 1114-
1117
12. Lorenz RR, Solares CA, Williams P, Sikora J, Pelfrey CM, Hughes GB, Tuohy VK
13. Interferon-gamma product to inner ear antigens by T-cells from patients with
autoimmune sensorineural hearing loss, J.Neuroimmunol., 2002, 130(1-2): 173-178
14. Solares CA, Edling AE, Johnson JM, Baek MJ, Hirose K, Hughes GB, Tuohy VK,
Murine autoimmune hearing loss mediated by CD4+ T cells specific for inner ear
peptides, J. Clin. Invest. 113:1210–1217 (2004).
15. Pathak S, Goldofsky E, Vivas EX, Bonagura VR, Vambutas A, IL-1β is overexpressed
and aberrantly regulated in corticosteroid no9nresponders with autoimmune inner ear
disease, J.Immunol, 2011, 186(3): 1870-1879
16. Vambutas A, De Voti J, Goldofsky R, Gordon M, Lesser M, Bonagura V, Alternate
splicing of interleukin-1 receptor type II (IL1R2) in vitro correlates with clinical
glucocorticoid responsiveness in patients with AIED. PLoS ONE, 2009, 4:e5293
17. RenJ, Li H, Lu Y, et al, Determinetion of tumor necrosis factor and interleukin 6 in
serum of patients with sudden sensorineural hearing loss, Lin Chuang Er Bi Yan Hou
Ke Za Zi, 1998, 12:311-313

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18. Suslu N, Yilmaz T, Gursel B, Utility of anti-HSP 70, TNF-α, ESR, antinuclear antibody
and antiphospholipid antibodies in the diagnosis and treatment of sudden
sensorineural hearing loss, The Laryngoscope, 2009, 119: 341-346
19. Loveman DM, de Comarmond C, Cepero R, Baldwin DM, Autoimmune sensorineurl
hearing loss: clinical course and treatment outcome, Semin.Arthritis Rheum, 2004,
34(2): 538-43
20. Niparko JK, Wang NY, Rauch SD, Russell GB, Espeland MA, Pierce JJ, Bowditch S,
Masuda A, Gulya AJ, Gantz BJ, Hughes GB, Brookhauser PE, Hannley MT, Telian
SA, the AIED study group, Harris JP, Serial audiometry in a clinical trial of AIED
treatment, Otol.Neurotol, 2005, 26(5): 908-917
21. Harris JP, Weisman MH, Derebery JM.. Espeland MA, et al,Treatment of
Corticosteroid-Responsive Autoimmune Inner Ear Disease With Methotrexate: A
Randomized Controlled Trial, JAMA. 2003; 290(14):1875-1883
22. Timothy C.Hain, Autoimmune inner ear disease, (AIED), http: www.dizziness-and-
balance.com/disorders/autoimmune/aied.html
23. Buniel MC, Geelan-Hansen K, Weber PC, Tuohy VK , Immunosuppressive therapy
for autoimmune inner ear disease, Immunotherapy, 2009; 1(3): 425–434.
24. Bovo R, Ciorba A, Martini The diagnosis of autoimmune inner ear disease: evidence
and critical pitfalls, Eur Arch Otorhinolaryngol. 2009 Jan; 266(1):37-40. doi:
10.1007/s00405-008-0801-y. Epub 2008 Sep 6
25. Broughton SS, Meyerhoff WE, Cohen SB. Immune-mediated inner ear disease: 10-
year experience.Semin Arthritis Rheum. 2004; 34: 544–548.
26. Bovo R, Aimoni C, Martini A. Immune-mediated inner ear disease. Acta
Otolaryngol.2006; 126: 1012–1021

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